Science - Adh7950 SM

Supplementary Materials for
DNA damage induces p53-independent apoptosis through ribosome stalling
Nicolaas J. Boon et al.
Corresponding authors: Reuven Agami, r.agami@nki.nl; Thijn R. Brummelkamp, t.brummelkamp@nki.nl
Science 384, 785 (2024)

DOI: 10.1126/science.adh7950
The PDF file includes:
Materials and Methods

Figs. S1 to S21
Table S6
References
Other Supplementary Material for this manuscript includes the following:
Tables S1 to S5
MDAR Reproducibility Checklist
31 Materials and Methods
32
33 Cell Culture, Reagents and Antibodies
34 HAP1 (CVCL_Y019) and KBM7 (CVCL_A426) cells were cultured in Iscove’s Modified
35 Dulbecco’s Medium (IMDM) (Thermo Fisher Scientific) supplemented with 10% heat-
36 inactivated fetal calf serum (FCS, Capricorn Scientific), GlutaMAX (Gibco), and penicillin-
37 streptomycin (P/S, Gibco) at 37°C in 5% CO2. PC3 (CRL-1435) cells were cultured using
38 RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% FCS, GlutaMAX, and
39 P/S at 37°C in 5% CO2. A549 (CCL-185) and 293T (CRL-3216) cells were cultured in
40 Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) supplemented with
41 10% FCS, GlutaMAX, and P/S at 37°C in 5% CO2. Cell lines used in this study were regularly
42 tested for mycoplasma. Reagents used in this study were DMSO (Sigma), etoposide (Sigma,
43 0.16-25μM), hydroxyurea (Sigma, 2mM), cisplatin (Sigma, 2.5μM), anisomycin (Sigma,
44 5μg/ml), camptothecin (Sigma, 1μM), neocarzinostatin (Sigma, 500ng/ml), puromycin (2
45 μg/ml), Z-VAD-FMK (Apex Bio Technology 100μM), VE-821 (Sigma, 10 μM), KU-60019
46 (Sigma, 10 μM), Imatinib/Gleevec (Sigma, 10 μM), doxycycline (Sigma, 1μg/ml),
47 cyclohexamide (CHX, Sigma, 100μg/ml), EdU (Invitrogen, 20 μM), or interferon-gamma
48 (Sigma, 5-20ng/μl). Antibodies used in this study were anti-a-tubulin (Thermo Fisher
49 Scientific MA1-80017), anti-SLFN11 (Santa Cruz 515071), anti-p53 (Santa Cruz 126), anti-
50 ZAKa (Bethyl A301-993A), anti-γH2AX (Merck 05-636), anti-Cleaved Caspase 3 (For
51 immunostaining, BD Biosciences 559565), anti-Caspase 3 (For immunoblot, Cell Signaling
52 Technology 9662), anti-Cleaved PARP (Cell Signaling Technology 9541), anti-puromycin
53 (Merck MABE343), anti-GCN2 (Cell Signaling Technology 3392), anti-p-JNK (Cell Signaling
54 Technology 4668), anti-p-p38 (Cell Signaling Technology 4511), anti-p-ERK1/2 (Cell
55 Signaling Technology 4695), anti-p-MAP2K4 (Cell Signaling Technology 4514), anti-p-
56 CHK1 (Cell Signaling Technology 2348), anti-p-eIF2a (Abcam 32157), anti-p-KAP1 (Bethyl
57 A300-767A), anti-ATR (Santa Cruz 515173), anti-ATM (Cell Signaling Technology 2873S),
58 anti-Lamin B1 (Cell Signaling Technology 12568), anti-CD3 (BD Biosciences 550368), anti-
59 CD8 (Biolegend 317409), anti-CD4 (BD biosciences 555369).
60
61 Organoid culture
62 The collection of colorectal tissue for the generation of CRC organoids has been performed
63 according to the guidelines of the European Network of Research Ethics Committees (EUREC)
64 following European, national, and local law. In all cases, patients signed informed consent after
65 ethical committees approved the study protocols. CRC PDOs p6T, p19bT and p26T were
66 obtained from previous studies (35) and were cultured as described (35, 41). Briefly, organoids
67 were cultured in Basement Membrane Extract (BME; R&D systems) droplets and expanded in
68 DMEM/F12 (Invitrogen) with 1% P/S (P/S, Lonza), 1% Hepes buffer (Invitrogen) and 1%
69 Glutamax (Invitrogen), 20% R-spondin conditioned medium, 10% Noggin conditioned
70 medium, 1x B27 (Gibco), 1.25mM n-Acetyl Cysteine (Sigma), 10mM Nicotinamide (Sigma),
71 50ng/ml EGF (Peprotech), 500nM A83-01 (Tocris), 10mM SB202190 (Gentaur). CRC PDO
72 lines OPT379-0000014 (OPTIC14) and RAS11 were obtained from UMC Utrecht, cultured in
73 BME droplets (R&D Systems) and expanded in DMEM/F12 (Invitrogen) with 1% P/S (P/S,
74 Lonza), 1% Hepes buffer (Invitrogen) and 1% Glutamax (Invitrogen), supplemented with 20%
75 Rspondin1 conditioned medium, 10% Noggin conditioned medium, 1x B27 (Gibco), 1.25 mM
76 N-Acetyl-L-Cysteine (Sigma), 500 nM A 83-01 (Tocris), 0.5 nM Wnt Surrogate-Fc Fusion
77 protein (U-protein express), 50 ng/ml rc human EGF (Peprotech), 50 ng/ml human FGF-basic
78 (Peprotech), 100 ng/ml rc human IGF1 (Biolegend), 10 μM Y-27632 (Gentauer) at 37°C / 5%
79 CO2. Organoids were passaged using Trypsin-EDTA (Sigma-Aldrich). Trypsin activity was
80 abrogated using 0.5mg/ml soybean trypsin inhibitor (Sigma-Aldrich). Residual trypsin was
81 washed out using Advanced DMEM/F12, the cells were resuspended in BME and culture
82 medium was supplemented for the first two days with 10µM Y-27632 dihydrochloride
83 (Gentaur).
84
85 Primary human T cell culture
86 Peripheral blood mononuclear cells (PBMC) were isolated from anonymized healthy donors
87 using Ficoll Paque Plus (Cytiva) density gradient separation and stored in liquid nitrogen until
88 further use. PBMCs were then cultured in RPMI 1640 medium (Life Technologies)
89 supplemented with 10% human serum (Thermo Fisher Scientific) and 1% P/S (Thermo Fisher
90 Scientific). A CD3+ peripheral blood lymphocytes (PBL) positive fraction was obtained from
91 PBMC by stimulation with 100 IU/ml IL-2 (Proleukin) and CD3/CD28 dynabeads (CTS) for
92 3 days. T cell purity was subsequently measured using live-cell flow cytometry and the cells
93 were used for further experiments when purity was >85%.
94
95 Plasmids and cloning
96 Single guide RNAs (sgRNAs) were generated using the Broad Institute’s CRISPick platform
97 (51). They were purchased as ssDNA oligo’s, annealed and cloned into PX330 or
98 pLentiCRISPRv2 (with puromycin or blasticidin as selection markers) digested with
99 respectively BbsI or BsmBI (New England Biolabs). sgRNAs were generated for TP53
100 (GTTGCAAACCAGACCTCAGG, exon 5), SLFN11 (52) (sg1:
101 TACACTGGTCTGCTAAGGGG, exon 2 & sg2: CCTTTTCACAAAATTCACAA, exon 1),
102 GCN2 (TATATGTAAAAGTGGATTTG, exon 1), and ZAKa
103 (GAAGTGAATGATATGCCCCT, exon 14). A non-targeting control sgRNA was used with a
104 sequence targeting the zebrafish TIA gene (GGTATGTCGGGAACCTCTCC). Genetic
105 modification was confirmed by PCR with primers designed for the corresponding genomic
106 DNA sequence.
107
108 The SLFN11 coding sequence was amplified by PCR to generate AgeI and NheI overhang sites
109 from a cDNA ORF plasmid (HG29932-UT Sino Biological), digested and subsequently cloned
110 into the pCW57.1 backbone containing a blasticidin resistance cassette. The mutations E209A
111 and K652D were generated by site directed mutagenesis and confirmed by sanger sequencing.
112
113 PCR primers used in this study were:
114
Gene Forward Reverse
TP53 CACATGACGGAGGTTGTGAG GATGGAATCTCGCTCTGTCG
SLFN11 CTGAAGATCGCTCTGTCAAGC CAAATGCAGGGACGTATTCTG
exon 1
SLFN11 GGCAACCTTGATATGC CTGATGATCAGCCAGATTTGG
exon 2
GCN2 GTATCAGCTGGTTGGGACTGTGTCTG GGAATGGGAGCAGGAAGGAGAAAATAC
ZAKa GATTCCTTCTGTCTGTTGCTCTG CTAACACGTGATCTGCTGCATAGTAGG
115
116 Generation of Cell Lines
117 Cell lines were generated by PX330 transfection or lentiviral transduction. HAP1 cells were
118 transfected with PX330 containing the sgRNA of choice along with a plasmid containing a
119 constitutively expressed blasticidin resistance cassette with Xfect according to manufacturer’s
120 instructions. Transfected cells were selected with 40μg/ml blasticidin for 48 hours and allowed
121 to recover for 24 hours in fresh medium before seeding single cells into a 96-wells plate.
122 Genetic modification of individual clones was measured by PCR and Sanger sequencing.
123 Abrogation of the protein was confirmed by immunoblot analysis.
124
125 Polyclonal knockout pools were generated by lentiviral transduction with lentiCRISPRv2
126 containing a blasticidin or puromycin resistance cassette along with the sgRNA of choice.
127 Expression of SLFN11 was reintroduced in SLFN11-deficient HAP1 and 293T cells by
128 lentiviral transduction with pCW57.1-blast. Lentivirus was produced in 293T cells transfected
129 with VSVG, ΔVPR, pAdvantage and a lentiviral plasmid (lentiCRISPRv2 or pCW57.1-blast).
130 The supernatant containing virus was collected two days after transfection and passed through
131 a 40μm filter. The virus was applied to HAP1, 293T, A549 or PC3 cells along with 8μg/ml
132 protamine sulfate for 24 hours and recovered for 24 hours in fresh medium. Cells were then
133 selected with blasticidin (40μg/ml for HAP1, 70μg/ml for 293T, and 100μg/ml for A549, and
134 10μg/ml for PC3 cells) or puromycin (1 μg/ml for HAP1 and A549) for 48 to 120 hours and
135 cultured for 6 more days before use in experiments. (Partial) abrogation or overexpression of
136 proteins was measured by immunoblot analysis and flow cytometry.
137
138 Base editing of endogenous SLFN11
139 HAP1 cells were treated with 1µM Nedisertib (Selleckchem) 2 hours prior to transfection to
140 prevent non-homologous end joining and promote homologous recombination (HR). HAP1
141 cells were then transfected as described in “Generation of Cell Lines” with PX330 containing
142 a guide that spans codon 209 (ACTGAGACTCAGGAAAAGGC). Additionally, a donor oligo
143 was co-transfected containing the sequence to mutate the glutamic acid on position 209 to an
144 alanine (E209A) by HR. The following day cells were passaged to a 10cm dish with media
145 supplemented with 1µM Nedisertib (Selleckchem). The selection and subsequent steps were
146 performed according to “Generation of Cell Lines". The endogenous mutation was confirmed
147 by PCR and sanger sequencing (forward primer: CTGAAGATCGCTCTGTCAAGC, reverse
148 primer: CAAATGCAGGGACGTATTCTG). Donor oligo (codon 209 in bold and underlined):
149 aattttccaaaaagactatcttgaatatggtgaaatcctgccttttcctgcgtctcagttagtagagtttaaacagttctctacaaaacacttcc
150 aagaa.
151
152 SDS-PAGE and Immunoblot Analysis
153 To visualize global translation, puromycin incorporation (53) was performed by treating cells
154 with medium containing puromycin (2μg/ml) for 10 minutes before washing once and
155 advancing to cell lysis. Cells were lysed directly in 2x LD Sample buffer (Invitrogen) with
156 100μM DTT, sonicated, and boiled for 10 minutes at 100 °C. Organoids were incubated with
157 10μg/ml puromycin for 10 minutes, placed on ice and washed with ice-cold D-PBS. Next,
158 organoids were dislodged in PBS-10mM EDTA (containing protease and phosphatase
159 inhibitors) on ice and incubated for another 15 minutes in this mixture (rotating in 15ml tubes
160 at 4°C) to dissolve the BME. Upon centrifugation, organoid pellets were lysed as described
161 above. Proteins were resolved by SDS-Page and transferred onto nitrocellulose membranes
162 (Amershan Protan, 0.45μM pore size). These membranes were blocked in 5% milk in TBST
163 (TBS with 0.1% Tween) before primary antibody staining in 3% BSA in TBST for 16 hours at
164 4 °C. Secondary staining with HRP (Thermo Fisher Scientific) was performed for 1 hour at RT
165 in blocking buffer. The HRP signal was measured using Clarity Western 306 ECL substrate
166 (Bio-Rad) or SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific)
167 and imaged on a Universal Hood II Gel Doc system (Bio-Rad).
168
169 Phos-tag gel electrophoresis
170 Phos-tag gel electrophoresis was performed by resolving lysed samples with 7% SDS-PAGE
171 containing 10.7 to 21.4μM Phos-tag (WAKO) and MnCl2 (Sigma). The protein gel was rinsed
172 with transfer buffer containing 10mM EDTA three times then washed with transfer buffer once
173 before proceeding with the transfer onto nitrocellulose membranes (Amershan Protan, 0.45μM
174 pore size) according to manufacturer’s instructions (WAKO). The membrane blots were
175 subsequently processed as described in ‘SDS-PAGE and immunoblot analysis’.
176
177 Immunofluorescence and confocal microscopy
178 HAP1 cells were grown on a cover slip in a 24-wells plate and fixed with Fix buffer I (BD
179 Biosciences) for 10 minutes at 37 °C, followed by permeabilization with Perm Buffer III (BD
180 Biosciences) on ice for 30 minutes. Blocking was performed with PBS with 10% FCS for 30
181 minutes at RT before incubation with the primary antibody in blocking buffer for 1h at RT.
182 Finally, the cells were washed once more and incubated with one or multiple secondary
183 fluorescent antibodies (Alexa Fluor 488, 568 or 647 conjugated to anti-mouse or anti-rabbit)
184 along with DAPI for 1 hour at RT. The cover slip was mounted on a slide with Aqua-
185 Poly/Mount (Polysciences) before imaging with the confocal scanner SP5 (Leica). Files were
186 exported as .TIFF or .lif and processed with Adobe Photoshop or ImageJ and Adobe Illustrator.
187
188 Organoid immunofluorescence and confocal microscopy
189 Organoids were seeded in chambered coverslips (Ibidi) and treated with etoposide or DMSO
190 for 18 hours. They were then treated with 10μg/ml puromycin for 10 minutes before washing
191 once with PBS for 5 minutes at 37 °C. The organoids were fixed with 4% PFA with 0.25%
192 glutaraldehyde for 20 minutes at RT. They were washed with PBS and incubated in blocking
193 buffer (0.3%TX100-PBS, 10%BSA) for 2 hours at RT. Next, they were incubated with the
194 primary antibody in blocking buffer O/N at 4 °C, washed with blocking buffer several times
195 and incubated O/N at 4°C with the secondary antibody (Alexa Fluor 488, 568 or 647 conjugated
196 to anti-mouse or anti-rabbit) along with DAPI in blocking buffer. They were washed twice
197 before directly imaging the chambered coverslips with the confocal scanner SP5. Files were
198 exported as .lif and processed with ImageJ and Adobe Illustrator.
199
200 Incucyte live-cell imaging
201 Cells were plated in a 96-well black/clear bottom plate and treated the following day for 0 to
202 72 hours with etoposide or Incucyte Annexin V red dye (Sartorius) or both. Images were
203 collected every 4 hours by the Incucyte ZOOM (Sartorius). The images were analysed by the
204 Incucyte ZOOM Live-cell Analysis System to generate the cell confluence (%) and Annexin
205 V signal (%) data. The Annexin V signal (%) was normalized to the cell confluence (%) by
206 Microsoft Excel and subsequently processed with GraphPad Prism and Adobe Illustrator. Data
207 are shown as mean ± SD (n=3, technical replicates).
208
209 Flow cytometry analysis
210 Puromycin incorporation was performed as described in ‘SDS-PAGE and immunoblot
211 analysis’ before advancing to cell collection. HAP1, A549 and PC3 cells grown in 6cm plates
212 were collected by trypsinization or T cells grown in 6-wells plates by centrifugation and fixed
213 in Fix buffer I for 10 minutes at 37 °C. The cells were subsequently permeabilized with Perm
214 Buffer III on ice for 30 minutes and blocked in PBS with 10% FCS for 30 minutes at RT. The
215 cells were then incubated with primary antibodies in blocking buffer for 1 hour at RT followed
216 by incubation with one or multiple secondary fluorescent antibodies (Alexa Fluor 488, 568 or
217 647 conjugated to anti-mouse or anti-rabbit) along with DAPI in blocking buffer for 1 hour at
218 RT. Cells were passed through a 40μM filter to dissociate any cell clumps before analysis by
219 an LSRFortessa Cell Analyser (BD biosciences). Because the fraction of ‘high translation’ cells
220 influences the puromycin staining intensity (fig. S4A) (most likely due to the inability to reach
221 saturating conditions for puromycin signal), the laser intensity was adjusted between
222 measurements to normalize the samples on the MFI of the high translation population. Files
223 were exported as .fcs and processed with FlowJo followed by Adobe Illustrator. An equal
224 number of cells is displayed in each FACS plot, except the plots where SLFN11 positivity was
225 gated for which may show fewer cells.
226
227 DNA fragmentation labeling by TUNEL
228 Staining of DNA fragmentation was performed with the Click-iT Plus TUNEL assay kit
229 (Thermo Fisher Scientific). Cells were collected, fixed and permeabilized as previously
230 described in “Flow cytometry analysis”. Afterwards, 2E5 cells in suspension were submitted
231 to the TdT reaction and the Click-iT Plus reaction as described in the manufacturer’s
232 instructions of the Click-iT Plus TUNEL assay kit (Thermo Fisher Scientific, C10617), using
233 the volumes of each component described for 1 coverslip. The cells were then blocked with
234 3% BSA in PBS for 30 min at RT before proceeding with antibody staining according to “Flow
235 Cytometry analysis” using 3% BSA as the blocking buffer.
236
237 EdU labeling of highly replicating cells.
238 Staining of highly replicating cells was performed by EdU incorporation and click chemistry.
239 HAP1 cells were incubated with 20 μM EdU (Invitrogen) simultaneously with etoposide
240 treatment. Cells were then harvested, fixed and stained according to “Flow Cytometry analysis”
241 before proceeding with EdU labeling. The EdU labeling was performed by incubating the
242 samples with 100mM Tris-HCl (pH 8.5), 4 mM CuSO4, 10 mM ascorbic acid, and 10 μM
243 Fluor-Azide 488 (1:1000; ATTO) for 30 min.
244
245 Cell viability analysis
246 HAP1 or KBM7 cells were plated in 96-well plates and treated the following day for 0 to 72
247 hours. CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, G7570) was used
248 according to manufacturer’s instructions to determine the viability of cells and the
249 luminescence was measured by the microplate reader PHERAstar FS. Data are shown as mean
250 ± SD (n=3, technical replicates).
251
252 Cell fractionation
253 Nuclear and cytoplasmic native protein and RNA were isolated from the same experimental
254 samples for Immunoblot and RT-PCR analysis using the PARIS kit (Thermo Fisher Scientific)
255 according to manufacturer’s instructions.
256
257 Real time PCR analysis of tRNA abundance
258 HAP1, PC3 and A549 cells were cultured in 6cm dishes while T cells were cultured in 6-wells
259 plates. The adherent cells were collected by trypsinization, the T cells by centrifugation and
260 the organoids were dislodged as described in ‘Sample preparation for patient-derived colon
261 organoids’ before total RNA extraction using the RNeasy mini kit (Qiagen) and QIAshredder
262 (Qiagen) according to manufacturer’s instructions. DNAse treatment was subsequently
263 performed with the RNase-Free DNase set (Qiagen). Total RNA was demethylated to improve
264 reverse transcription of tRNAs before cDNA synthesis using the rtStar tRNA Pretreatment &
265 First-Strand cDNA synthesis kit (Arraystar) with 5μg of input RNA. The synthesized cDNA
266 was used for RT-PCR analysis of U6 snRNA (5’-GCTTCGGCAGCACATATACTAAAAT-
267 3’ and 5’-CGCTTCACGAATTTGCGTGTCAT-3’), tRNA-UAA 1-1 (5’-
268 ACCAGGATGGCCGAG-3’ and 5’- TACCAGGAGTGGGGTTC-3’), and tRNA-UAA 3-1
269 (5’-ACCAGAATGGCCGAG-3’ and 5’-TACCAGAAGTGGGGTTC-3’). Data are shown as
270 mean ± SD (n=3, technical replicates). P-values were calculated using a two-tailed t-test,
271 ns=not significant, *p<0.05, **p<0.005. The RT-PCR analysis was performed with iQ SYBR
272 Green Supermix (Bio-Rad) and analysed in a QuantStudio 6 Flex Real-Time PCR system
273 (Thermo Fisher Scientific).
274
275 Haploid genetic screens
276 Haploid genetic screens were performed as described by Brockmann et al (22). Virus was
277 produced by transfecting 293T Cells with GT-GFP, VSVG, ΔVPR, and pAdvantage. 48 hours
278 post transfection the viral supernatant was collected, passed through a 40μM filter and
279 concentrated with Amicon filters according to manufacturer’s instructions. Medium was re-
280 applied to the 293T cells and this procedure was repeated after 24 hours. The concentrated
281 virus was immediately applied to HAP1 cells with 8μg/ml Protamine Sulfate. After 24 hours
282 the cells were recovered in fresh medium and cultured as described above for 8-10 days before
283 use in genetic screens.
284
285 To perform the genetic screens, the cells were expanded to 3E9 cells and treated with DMSO,
286 etoposide or anisomycin. For the translation screens, all cells were subsequently treated with
287 puromycin (2μg/ml) for 15 minutes before removing the medium and washing the cells with
288 PBS once. Next, the cells were harvested by trypsinization from T175 flasks, washed once with
289 PBS, fixed, permeabilized, and blocked as described in the ‘flow cytometry analysis’ methods
290 section followed by primary antibody staining for puromycin, cleaved caspase-3 or p-JNK and
291 DAPI. The cells were passed through a 40μm filter before being sorted on a FACSAria Fusion
292 cell sorter (BD Biosciences) gating for haploid G1 cells by DAPI and collecting 10E6 cells for
293 the high and low population, which represent the cell populations with respectively the 5%
294 highest and lowest fluorescent signal per molecular phenotype (puromycin, cleaved caspase-3,
295 or p-JNK). Genomic DNA isolation, sequencing library preparation and sequencing analysis
296 was performed as described previously (22). Reads were aligned to the reference genome
297 (GRCh38), allowing one mismatch, and the sense insertions in the high and low populations
298 were compared using a two-sided Fisher’s exact test to determine the significant differences
299 (FDR corrected p<0.05). The analysis was processed using GraphPad Prism software and
300 Adobe Illustrator to generate fishtail plots.
301
302 Ribosome profiling
303 Sample preparation for ribosome profiling
304 The construction of RPF libraries was done as previously described (54), with no addition of
305 harringtonine treatment. Total RNA was isolated using Trizol reagent (Invitrogen), according
306 to manufacturer’s instructions and rapid gel extraction was used followed by overnight
307 precipitation at −20 °C for RNA extraction from the polyacrylamide gels. rRNA depletion was
308 not performed and rRNA was removed during the data processing. The pooled library was
309 analysed on a 2100 Bioanalyser using a 7500 chip (Agilent, Santa Clara, CA), quantified by
310 qPCR (Cat.no. 07960140001, Roche, Switzerland) and subsequently diluted to 10nM. Next,
311 the library was loaded with 1.5pM on a NextSeq550Dx system (RUO mode) and sequenced
312 with 75-base single-read using a 75 Cycles High Output Kit v2.5 (Illumina Inc., San Diego).
313
314 Sample preparation for patient-derived colon organoids
315 Six-day old organoids (grown from single cells) were dislodged from BME using 1 mg/ml
316 dispase II (Thermo Fisher Scientific) for 15 min at 37˚C. Organoids were mechanically
317 dissociated by pipetting, washed extensively with advanced DMEM/F12 medium and then
318 seeded in suspension culture plates (Greiner Bio-One) in CRC medium containing 7.5% BME.
319 Three hours after seeding, organoids were treated for 18 hours with etoposide or DMSO. To
320 harvest the cells, the organoids were treated with 100μg/ml CHX in warm medium for 5
321 minutes, were washed with ice-cold PBS containing 100μg/ml CHX and were then dislodged
322 in PBS+CHX until all organoids were collected. The organoids were centrifuged for 5 minutes
323 at 1500rpm and washed once with PBS+CHX before proceeding with lysis as described in
324 ‘Sample preparation for ribosome profiling’.
325
326 Alignment
327 Adaptors were removed from FASTQ files using cutadapt (55) with parameters (-quality-
328 base=33 -O 7 -e 0.15 -m 20 -q 5). Ribosomal RNA (rRNA) and tRNA contaminants were
329 removed by aligning FASTQ reads with Bowtie2 (56) and parameters (-seed 42 -p1 -local)
330 against a reference library (rRNA reference is obtained from GENCODE v19: rRNA,
331 MT_RNA, rRNA_pseudogene, and tRNA reference data is obtained from GtRNAdb (57).
332 Preprocessed FASTQ files are then aligned with TopHat2 (58) and Bowtie2 against
333 GRCh37/hg19 and GENCODE v19/BASIC transcripts with Ensembl coordinates using the
334 parameters (seed 41 -n 2-m 1 -novel-juncs -- no-novel-indels –no-coverage-search –segment-
335 length 25). Aligned reads were then filtered for minimum mapping quality of 10. Quality
336 control of frames and periodicity of RPFs were performed using the RiboWaltz package.
337
338 Diricore analysis
339 During subsequence analysis, codon occupancy frequency of RPFs is compared between two
340 tested conditions (e.g. etoposide treated versus control), as previously described(21). RPF
341 density analysis is performed by comparison of normalized 5’-RPF densities per codon
342 between two conditions. For transcript specific Diricore subsequence plots, transcripts with
343 and without the codon of interest were selected (based on gencode sequence). Codon
344 occupancy per transcript was obtained from RiboWaltz (59). Codon occupancies were added
345 up and a normalization by the entire number of codon reads per dataset was performed. The
346 codon normalized occupancy between two conditions was then compared and plotted as in the
347 aforementioned Diricore subsequence plots.
348
349 Code Availability
350 Analysis pipelines for haploid screens are available at
351 https://github.com/BrummelkampResearch. Analysis pipelines for ribosome profiling are
352 available at https://github.com/pkorner218/Ribosome_Diricore_pipeline/.
A
Cell viability (% of control) 150 HAP1 + Gleevec
KBM7 + Gleevec
100
50
0
0h 24h 48h 72h
353
354
355 Fig S1. BCR-ABL inhibition does not affect viability of HAP1 cells. Cell viability of HAP1
356 and KBM7 cells treated with Gleevec (10μM) was measured from 0 to 72 hours by
357 CellTiterGlo. The luminescence was plotted in % relative to the untreated control.
358
A B
Etoposide _ +
DNA ladder CC3 positive
size in bp TUNEL positive and CC3 positive
2000
1500 Untreated Etoposide
1200
1000 5
900 10
800
700 4 1.9% 42.9%
600 10
500
TUNEL
3
400 10
300 2
10
3.6% 49.5%
1
200 10
1 2 3 4 5 1 2 3 4 5
10 10 10 10 10 10 10 10 10 10
100
Cleaved caspase 3
C D 2μM etoposide in HAP1
0h 8h 16h 24h
100 WT
WT 0.5μM Untreated
Cell confluence (%)
75 WT 1μM
WT 2μM
50
Etoposide
25
0
0 24 48 72
Time in hours
359
360
361 Fig S2. HAP1 cells undergo DNA fragmentation and dose-dependent cell death in
362 response to DNA damage. (A) Six μg of gDNA was extracted from HAP1 cells treated with
363 etoposide (10μM) for 8 hours, run on an agarose gel and visualized by GelRed. (B) DNA
364 fragmentation (TUNEL) and caspase-3 cleavage were measured in flow cytometry in HAP1
365 cells treated with etoposide (10μM) for 8 hours. (C) Cell confluence (%) was determined in
366 HAP1 cells treated with several concentrations of etoposide with live-cell imaging by the
367 Incucyte ZOOM. The live-cell imaging was performed for 72 hours and pictures were taken
368 every 4 hours. (D) Representative images taken by the Incucyte ZOOM of HAP1 cells during
369 treatment with etoposide (2μM) at several timepoints.
370
A HAP1 A549 B Untreated Etoposide
WT ∆TP53 WT ∆TP53 p53 positive
9% CC3+
Normalized count
TP53 (short)
sg-p53
9.6%
TP53 (long)
p53-null
1 2 3 4 1 2 3 4
α-tubulin 10 10 10 10 10 10 10 10
Cleaved caspase 3
C D
A549
CC3 positive
Time in hours
Untreated Etoposide
Etoposide
_ 2H 4H
5
10
(magenta)
0.2% 12.5%
y-H2AX
4
10
3
sgNT
10
2
10
Puromycin
Cleaved caspase 3
(green)
1
10
0 50 100 150 200 250 0 50 100 150 200 250
5
10
4
0.5% 7.6%
(yellow)
10
CC3
sgTP53
3
10
2
10
Overlay
1
10
0 50 100 150 200 250 0 50 100 150 200 250
Cell size (FSC-A - a.u.)
E
Low translation & CC3 positive
Untreated Gleevec Etoposide Gleevec + etoposide

Cleaved Caspase 3
4
10 0.1% 0.2% 5.7% 5.6%
3
10
2
10
1
10
2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Translation
371
372 Fig. S3. HAP1 and A549 cells undergo p53-independent apoptosis. (A) Immunoblot
373 analysis visualizing p53 abundance in polyclonal pools of HAP1 and A549 cells that were
374 transduced with Cas9 and sgNT or an sgRNA targeting TP53. (B) Schematic depiction that
375 illustrates how cleaved caspase-3 was measured by flow cytometry in a polyclonal pool
376 containing WT and TP53-deficient HAP1 cells. (C) The polyclonal pools of A549 cells
377 mentioned in (A) were treated with etoposide (25μM) for 24 hours before puromycin
378 incorporation (2μg/ml) for 10 minutes and flow cytometry analysis. (D) HAP1 cells treated
379 with etoposide for 0, 2 or 4 hours followed by puromycin incorporation (2μg/ml) for 10 minutes
380 were subjected to immunostaining. (E) Global translation (x-axis) and caspase-3 cleavage (y-
381 axis) were measured by flow cytometry in HAP1 cells treated with Gleevec (10μM) and
382 etoposide (5μM) for 4 hours followed by puromycin incorporation (2μg/ml) for 10 minutes.
383
A B
Low translation High translation Low translation High translation
50% 50% 50% 50%
UNT 1:1 CHX Normalized count UNT 1:1 CHX
Normalized count
17.3% 82.7% 10.6% 89.4%
UNT 10:1 CHX UNT 10:1 CHX
63.9% 36.1% 90.6% 10.4%
UNT 1:10 CHX UNT 1:10 CHX
2 3 4 5 2 3 4 5
10 10 10 10 10 10 10 10
Translation Translation
384
385 Fig. S4. The proportion of cells with high/low translation affects puromycin staining
386 intensity and correction thereof. (A) WT HAP1 cells were treated with or without CHX
387 (100μg/ml) in warm medium for 5 minutes before puromycin incorporation (2μg/ml) for 10
388 minutes. The untreated and treated populations were subsequently mixed at a ratio of 1:1, 10:1
389 or 1:10 and used for flow cytometry analysis for the puromycin signal. (B) Adjustment of the
390 laser intensity between experiments to normalize the samples on the MFI of the high translation
391 population enables correct quantification of the populations with high and low translation
392 levels.
393
A B
CUA CUC CUG
2 2 2
1 1 1
0 0 0
Average transcript RPF density
7 1 1 1
Untreated 1
(intra-gene normalized)
untreated vs etoposide
6 Untreated 2 2
Relative read density

2 2
Treated 1
5 Treated 2 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60
4 Position in respect to codon Position in respect to codon Position in respect to codon
3
2
CUU UUA UUG
2 2 2
1
0 1 1 1
5’ UTR START STOP 3’ UTR
0 0 0
1 1 1
2 2 2
-30 -15 0 60 -30 -15 0 60 -30 -15 0 60
Position in respect to codon Position in respect to codon Position in respect to codon
C
Proteins containing UUA Proteins lacking UUA

6
Untreated 1 Untreated 1
5 Untreated 2
5
Untreated 2
Treated 1 Treated 1
Treated 2 4 Treated 2
4
3
3
2
2
1
1
0 0
5’ UTR START STOP 3’ UTR 5’ UTR START STOP 3’ UTR
D
Proteins containing UUA
0.5
Amino Acid
ALA
UUA ARG
ASN
0.25 ASP
CYS
GLN
GLU
Fraction Readcounts
GLY
0 HIS
ILE
LEU
LYS
Proteins lacking UUA MET
PHE
0.5
PRO
SER
START_ATG
STOP
0.25 THR
TRP
TYR
VAL
394 0
395 Fig. S5. Ribosome profiling demonstrates global translation initiation stalling and UUA
396 specific leucine stalling. (A) Metagene density profiles depicting global shifts of RPFs to the
397 start of the coding sequence comparing two independent replicates of both etoposide treated
398 and untreated HAP1 cells. The y-axis is intra-gene normalized RPF density. (B) Diricore
399 analysis line plots depicting differential (etoposide/untreated) ribosome occupancy (5’-RPF) at
400 -30 to +60 of the leucine codons in control versus etoposide treated HAP1 cells. (C) The data
401 presented in fig. S5A was re-analysed for the proteins containing and lacking UUA codons.
402 Metagene density profiles depicting global shifts of RPFs to the start of the coding sequence
403 comparing two independent replicates of both etoposide treated and untreated HAP1 cells. The
404 y-axis is intra-gene normalized RPF density. (D) The data presented in Fig. 1G was re-analysed
405 for the proteins containing and lacking UUA codons. Diricore analysis bar plots depicting
406 differential codon usage (at position -15 of the RPFs) in etoposide treated versus untreated
407 HAP1 cells. The x-axis is the fraction of reads showing stalling per codon (adding up to 100).
408
A B
Leucine
UAA 3-1
Cytoplasmic Nuclear
1.5
Etoposide - + - + * *
Relative tRNA level

α-tubulin
1.0
Lamin B
0.5
Ponceau
0.0
Etoposide - + - +
Cytoplasm Nuclear
409
410 Fig S6. HAP1 cells show downregulation of leucine tRNA-UAA 3-1 in both the nuclear
411 and cytosolic fractions. (A) HAP1 cells treated with etoposide (10μM) in the presence of Z-
412 VAD-FMK (100μM) for 8 hours were used for nuclear/cytosolic fractionation and subjected
413 to immunoblot analysis. (B) Quantitative real time PCR analysis of leucine tRNA-UAA 3-1,
414 normalized to U6 snRNA, in both the nuclear and cytosolic fraction of HAP1 cells from fig.
415 6A.
416
A B
Translation (puromycin )
4
GCN2
3 GCN1 GCN2
gene rank (10 )

SLFN11
3
-1
SLFN11
log [MI]
-2
2
-3
-4
-5
-1 0 1 2 3 4 5 -4 -2 0 2 4
log 10 [total insertions] LOG2 ∆MI
C
SLFN12L SLFN13
SLFN14 SLFN12 SLFN5 SLFN11

Expression level
high
low
0 5 10 15 20
3
Gene rank (10 )
D
Insertions
1kb
High 95
SLFN5
Low 85
1kb
High 450
SLFN11
Low 95
1kb
High 29
SLFN12
Low 21
1kb
High 14
SLFN12L
Low 13
1kb
High 25
SLFN13
Low 11
1kb
High 2
SLFN14
Low 3
417
418 Fig. S7. SLFN11 is critical for inhibition of translation and ribosome stalling after DNA
419 damage. (A) Haploid genetic screen for translation in untreated cells as measured by
420 puromycin incorporation (2μg/ml) for 10 minutes and flow cytometry presented as a fishtail
421 plot. Genes are plotted according to their mutational index (MI) (y-axis) and the total number
422 of gene-trap insertions (x-axis) with positive and negative regulators in respectively blue and
423 orange. (B) Difference in mutational index (log2) between the puromycin screen treated with
424 etoposide versus untreated for every gene with at least 100 insertions in both screens. (C)
425 Expression levels of the human SLFN proteins in HAP1 cells as measured by RNA sequencing
426 (17). Low and high represent the lowest and highest 25% of expressed proteins. (D) Gene-trap
427 insertion plot of several gene locuses in the etoposide treated translation screen. Unique gene-
428 trap insertions (mutations) mapped to several loci in the low or high FACS-sorted channel are
429 shown for the screen. Solely insertions in the gene body between the start and stop codon in
430 the sense orientation were counted for the mutational index (MI).
431
A B C
ZAK KO
WT ∆SLFN11 WT ∆SLFN11 ATRi WT ∆SLFN11 WT ∆SLFN11
_ + _ + _ _ _ + _ _ _ + Etoposide _ _ _ _ +
Etoposide ATRi
_ 3h 5h 5h _ 3h 5h 5h
ATMi + + +
3h 5h 5h _ + _ + _ _ +
Etoposide Etoposide +
total ATM
p-CHK1 (s345) p-KAP1(s824)
total ATR
α-tubulin α-tubulin α-tubulin
432
433
434 Fig S8. ATR/ATM abundance and ATR/ATM kinase activity are unaffected in SLFN11-
435 deficient HAP1 cells. (A) Wild-type and SLFN11-deficient HAP1 cells were treated with
436 etoposide (5μM) for 4 hours and subjected to immunoblot analysis. (B) Wild-type and SLFN11-
437 deficient HAP1 cells treated with the ATR inhibitor VE-821 (10μM) and etoposide (5μM) for
438 4 hours were subjected to immunoblot analysis. (C) Wild-type and SLFN11-deficient HAP1
439 cells treated with the ATM inhibitor KU-60019 (10μM) and etoposide (5μM) for 4 hours were
440 subjected to immunoblot analysis.
441
A B Inducible SLFN11 expression
in ∆SLFN11 HAP1
WT ∆SLFN11 WT E209A
Tyrosine Leucine Leucine Leucine Leucine Leucine

GUA UAA 1-1 UAA 3-1 UAA 3-1 UAA 3-1 UAA 3-1
1.5 ns 1.5 ns 1.5 ns 1.5 ns * ns
Relative tRNA level

Relative tRNA level
Relative tRNA level
Relative tRNA level

1.0 1.0 1.0 1.0
0.5 0.5 0.5 0.5
0 0 0 0.0
Etoposide - + - + - + Doxycycline - - + + + +
Etoposide - + - + - +
C D
SLFN11 negative
SLFN11 positive HAP1
- doxycycline + doxycycline
SLFN11 Untreated Etoposide Untreated Etoposide

negative cells
Normalized count
Normalized count
WT SLFN11
w/o
doxycycline
1 2 3 4 5 2 3 4 2 3 4 2 3 4 2 3 4
10 10 10 10 10 10 10 10 10 10 10 10 10 10
10 10 10
SLFN11
SLFN11 SLFN11
negative cells positive cells
E209A SLFN11
Normalized count
Normalized count
induce SLFN11
w/ doxycycline
for 16h
1 2 3 4 5
10 10 10 10 10 2 3 4 2 3 4 2 3 4 2 3 4
10 10 10 10 10 10 10 10 10 10 10 10
SLFN11
Translation
E F
SLFN11 negative
SLFN11 positive 293T
293T - doxycycline + doxycycline
Untreated Etoposide Untreated Etoposide

Normalized count
WT SLFN11
Etoposide _ +
SLFN11
2 3 4 2 3 4 2 3 4 2 3 4
10 10 10 10 10 10 10 10 10 10 10 10
Translation
(Puromycin)
E209A SLFN11
Normalized count
α-tubulin
2 3 4 2 3 4 2 3 4 2 3 4
10 10 10 10 10 10 10 10 10 10 10 10
Translation
442
443 Fig. S9 Expression of WT but not mutant SLFN11 (E209A) leads to inhibition of
444 translation in SLFN11-deficient HAP1 cells and 293T cells in response to DNA damage.
445 (A) Quantitative real time PCR analysis of the Type I tRNA Tyrosine as a control, UUA-
446 corresponding tRNAs-UAA 1-1 and 3-1 normalized to U6 snRNA in WT (Tyrosine) or
447 SLFN11-deficient HAP1 cells (UAA 1-1 and 3-1) treated with etoposide (10μM) in the
448 presence of Z-VAD-FMK (100μM) for 8 hours. (B) Quantitative real time PCR analysis of the
449 UUA-corresponding tRNA-UAA 3-1 normalized to U6 snRNA. This was performed in clones
450 of SLFN11-deficient HAP1 cells transduced with a lentivirus to enable doxycycline inducible
451 expression of WT or E209A SLFN11. The cells were treated with doxycycline (1μg/ml) for 16
452 hours followed by etoposide (10μM) in the presence of Z-VAD-FMK (100μM) for 8 hours.
453 (C) Schematic representation of the flow cytometry gating strategy for SLFN11 positivity used
454 in D and F. (D) Global translation was measured by flow cytometry in polyclonal pools of
455 SLFN11-deficient HAP1 cells transduced with a lentivirus to enable doxycycline inducible
456 expression of WT or E209A SLFN11. The cells were treated with or without doxycycline
457 (1μg/ml) for 16 hours, then treated with etoposide (5μM) for 4 hours followed by puromycin
458 incorporation (2μg/ml) for 10 minutes. The SLFN11 positive and negative cell populations
459 were compared within each distinct sample. (E) 293T cells were subjected to etoposide (25μM)
460 for 24 hours followed by puromycin incorporation (2μg/ml) for 10 minutes and were then used
461 for immunoblot analysis to visualize inhibition of translation and SLFN11 expression. (F)
462 Global translation was measured by flow cytometry in polyclonal pools of SLFN11-deficient
463 293T cells transduced with a lentivirus to enable doxycycline inducible expression of WT or
464 E209A SLFN11. The cells were treated with doxycycline (1μg/ml) for 16 hours, then treated
465 with etoposide (25μM) for 24 hours followed by puromycin incorporation (2μg/ml) for 10
466 minutes. The SLFN11 positive and negative cell populations were compared within each
467 distinct sample.
468
A B
Low translation ∆GCN2 HAP1
30
Untreated Etoposide Etoposide + Z-VAD-FMK WT
∆SLFN11
** * ** * *
4
10
Cleaved caspase 3
cleaved caspase 3
3 20
% Cells with
10
2
10
10
1 8.7% 21.7% 21.1%
10
2 3 4 2 3 4 2 3 4
10 10 10 10 10 10 10 10 10
Translation
ed
PT
tin
id
la
at
C
os
p
re
op
is
nt
C
Et
U
C D
Low translation & TUNEL positive
Untreated Etoposide
4
10 2.5% 24.2%
3
10
HAP1
WT
2
10
∆SLFN11
WT pool
1
10 SLFN11 (short)
TUNEL
SLFN11 (long)
4
10 5.4% 3.7% α-tubulin
3
10
∆SLFN11
2
10
1
10
2 3 4 2 3 4
10 10 10 10 10 10
Translation
E F
Etoposide Neocarzinostatin
25 WT 25 WT
∆SLFN11 ∆SLFN11
20 20
Luminescence (a.u.)
Luminescence (a.u.)
15 15
10 10
5 5
0 0
d
uM
uM
uM
uM
uM
uM
ed
l
m
/m
/m
/m
e
g/
g/
g/
at
at
ng
ng
ng
5
10
20
re
re
5n
0n
0n
62
2.
00
00
00
1.
nt
nt
12
25
50
0.
10
20
40
U
469
470 Fig S10. GCN2 is required for global inhibition of translation and SLFN11 for the
471 induction of cell death upon DNA damage. (A) Global translation (x-axis) and caspase-3
472 cleavage (y-axis) were measured by flow cytometry in parental GCN2-deficient cells treated
473 with etoposide (5μM) and Z-VAD-FMK (100μM) for 4 hours followed by puromycin
474 incorporation (2μg/ml) for 10 minutes. (B) Caspase-3 cleavage was measured in four
475 independent experiments with parental HAP1 and SLFN11-deficient cells treated with
476 etoposide (5μM) for 4 hours, NCS (500ng/ml) for 4 hours, CPT (1μM) for 4 hours, HU (2mM)
477 for 16 hours, or cisplatin (2.5μM) for 16 hours followed by puromycin incorporation (2μg/ml)
478 for 10 minutes and flow cytometry. P-values were calculated using a two-tailed t-test, *p<0.05,
479 **p<0.005, ***p<0.0005. Data are shown as mean ± SD. (C) Global translation (x-axis) and
480 TUNEL positivity (y-axis) were measured using flow cytometry in WT and SLFN11-deficient
481 HAP1 cells treated with etoposide (10μM) for 8 hours followed by puromycin incorporation
482 (2μg/ml) for 10 minutes. (D) Expression of SLFN11 in HAP1 cells transduced with Cas9 and
483 an sgRNA targeting sg1-SLFN11 was visualized by immunoblot analysis. (E) Cell viability
484 was measured in the HAP1 cell pools displayed in fig. S10D treated with several concentrations
485 of etoposide for 24 hours. (F) Cell viability was measured in the HAP1 cell pools displayed in
486 fig. S10D treated with several concentrations of NCS for 24 hours
487
A C
Low translation SLFN11 negative
CC3 positive SLFN11 positive
+ Z-VAD-FMK Untreated Etoposide 5ng/ul IFNy 20ng/ul IFNy

3.8% 1.1%
Normalized count
Untreated
3
10
2
10
1
5.1% 9.1%
10 2 3 4 2 3 4 2 3 4 2 3 4
2 3 4 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10
10 10 10 10 10 10
Translation
+ Z-VAD-FMK
Cleaved Caspase 3
27.7% 1.5%
Normalized count
5ng/μl IFNy
Normalized To Mode
10
2
10
27% 30.8%
1
10 2 3 4 2 3 4 2 3 4 2 3 4
2 3 4 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10
10 10 10 10 10 10
+ Z-VAD-FMK p-JNK
29.1% 1.2%
Normalized count
20ng/μl IFNy
3
10
2
10
28.6% 30%
1
10
2 3 4 2 3 4 1 2 3 1 2 3 1 2 3 1 2 3
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Puromycin Cleaved Caspase 3
B
SLFN11 SLFN11
negative cells positive cells
Normalized count
SLFN11 positive
sg-SLFN11
SLFN11-null
1 2 3 4 5
10 10 10 10 10
489 SLFN11
490 Fig S11. IFN-γ treatment leads to SLFN11-independent inhibition of translation and
491 apoptosis. (A) Global translation (x-axis) and caspase-3 cleavage (y-axis) were measured by
492 flow cytometry in WT HAP1 cells treated with IFN-γ for 30 hours and with or without Z-VAD-
493 FMK (100μM) followed by puromycin incorporation (2μg/ml) for 10 minutes. (B) Schematic
494 representation of the flow cytometry gating strategy for SLFN11 positivity used in fig. S11C.
495 (C) Global translation, p-JNK, and caspase-3 cleavage were measured by flow cytometry in
496 polyclonal pools of HAP1 cells that were transduced with Cas9 and sg2-SLFN11 treated with
497 etoposide (5μM) for 4 hours or IFN-γ for 30 hours.
A
Low translation
CC3 positive
Untreated 20J/m2 UV & 4h recovery
2.2% 25.8%
3
10
WT
2
10
Cleaved Caspase 3
12.3% 56.4%
1
10
2 3 4 2 3 4
10 10 10 10 10 10
2.2% 11.8%
3
∆SLFN11
10
2
10
12.7% 22.4%
1
10
2 3 4 2 3 4
10 10 10 10 10 10
Translation
498
499 Fig S12. UV radiation induces both SLFN11-dependent and SLFN11-independent
500 inhibition of translation and apoptosis. (A) Global translation (x-axis) and caspase-3
501 cleavage (y-axis) were measured by flow cytometry in WT HAP1 cells treated with UV
502 radiation (20J/m2) and recovered for 4 hours followed by puromycin incorporation (2μg/ml)
503 for 10 minutes.
504
A CC3 positive
Untreated Etoposide ATRi Etoposide + ATRi
10
4
0.4% 7.1% 1.7% 7.3%
3
10
WT
2
Cleaved Caspase 3
10
1
10
2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
10
4
0.6% 1.2% 1.4% 2%
∆SLFN11
3
10
2
10
1
10
2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Translation
B CC3 positive
Untreated Etoposide ATMi Etoposide + ATMi

4
10
0.7% 8.9% 0.9% 10.1%
3
10
WT
2
10
Cleaved Caspase 3
1
10
1 2 3 4 5 1 2 3 4 5 101 102 103 104 105 1 2 3 4 5

10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
4
10 2.8% 1% 1.9% 2.5%
∆SLFN11
3
10
2
10
1
10
1 2 3 4 5 1 2 3 4 5 101 102 103 104 105 1 2 3 4 5

10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Translation
C D Low translation & CC3 positive
Untreated Etoposide 24h Etoposide & ATRi 24h

WT HAP1 etoposide 10
5
WT HAP1 etoposide & ATRi 1.3% 19.5% 43.5%

4
10
∆SLFN11 etoposide
∆SLFN11 etoposide & ATRi 3
WT
10
2
10
Cleaved Caspase 3
120 10
1
Viability (% of control)
100
5
10
80
4 2.3% 1.4% 10.4%
10
60
∆SLFN11
3
10
40
2
10
20
1
10
0
1
0h 4h 8h 24h 10 10
2
10
3
10
4
10
5
10
1
10
2
10
3
10
4
10
5
10
1
10
2
10
3
10
4
10
5
Translation
505
506 Fig S13. SLFN11-deficient HAP1 cells are re-sensitized to DNA damage by ATR
507 inhibition and undergo apoptosis. (A) Global translation (x-axis) and caspase-3 cleavage (y-
508 axis) were measured by flow cytometry in HAP1 cells treated with the ATR inhibitor VE-821
509 (10μM) and etoposide (5μM) for 4 hours followed by puromycin incorporation (2μg/ml) for
510 10 minutes. (B) Global translation (x-axis) and caspase-3 cleavage (y-axis) were measured by
511 flow cytometry in HAP1 cells treated with the ATM inhibitor KU-60019 (10μM) and
513 (C) Cell viability was measured at various timepoints in WT and SLFN11-deficient HAP1 cells
514 treated with etoposide (5μM) for 24 hours or etoposide (5μM) and VE-821 (10μM) for 24
515 hours. The luminescence values were plotted as a percentage relative to the untreated condition.
516 (D) Global translation (x-axis) and caspase-3 cleavage (y-axis) were measured by flow
517 cytometry in WT and SLFN11-deficient HAP1 cells treated with etoposide (5μM) for 24 hours
518 or etoposide (5μM) and VE-821 (10μM) for 24 hours followed by puromycin incorporation
519 (2μg/ml) for 10 minutes.
520
A WT SLFN11 sequence Sanger sequencing clone 4 B
codon 209
E209A
codon 209 endogenous mutagenesis WT
SLFN11
∆SLFN11
E209A cl.13 cl. 3
cl. 4
GAG to GCG SLFN11
α-tubulin
C Low translation & CC3 positive

D
Untreated Etoposide
4
10
2.7% 8.5% Etoposide (24 hours)
WT cl. 13
3
10
1.0
2
10
(relaltive to control)
Luminescence
WT cl. 13
1
10 E209A
SLFN11 cl. 4
0.5
∆SLFN11 cl. 3
E209A SLFN11 cl. 4
Cleaved Caspase 3
4
10
2.7% 1.8%
3
10
0.0
ed
uM
uM
uM
uM
uM
2
10
at
25
10
re
62
2.
1.
nt
0.
U
1
10
4
E
10
∆SLFN11 cl. 3
1.1% 0.72%
3
10
SLFN11 WT E209A WT E209A
Doxycycline _ _ + +
2
10
SLFN11
1
10
10
2
10
3
10
4
10
2 3
10
4
10 α-tubulin
Translation
F
10
4 2.4% 2.2% 5.9% 16.9%
WT SLFN11
3
10
2
10
Cleaved Caspase 3
1
10
2 3 4 2 3 4 2 3 4 2 3 4
10 10 10 10 10 10 10 10 10 10 10 10
10
4 1.3% 1.2% 0.7% 0.9%
E209A SLFN11
3
10
2
10
1
10
2 3 4 2 3 4 2 3 4 2 3 4
10 10 10 10 10 10 10 10 10 10 10 10
Translation
521
522 Fig S14. SLFN11-E209A phenocopies SLFN11 deficiency. (A) Schematic representation of
523 the endogenous mutation of SLFN11 codon 209 from WT alanine (E) to aspartic acid (A) in
524 HAP1 cells. (B) Expression of SLFN11 in several HAP1 clones was visualized by immunoblot
525 analysis. (C) Global translation (x-axis) and caspase-3 cleavage (y-axis) were measured by
526 flow cytometry in the HAP1 clones displayed in fig. S14B treated with etoposide (5μM) for 4
527 hours followed by puromycin incorporation (2μg/ml) for 10 minutes. (D) Cell viability in the
528 HAP1 clones displayed in fig. S14B treated with various concentrations of etoposide for 24
529 hours. (E) Expression of SLFN11 in SLFN11-deficient HAP1 cells transduced with a lentivirus
530 to enable inducible expression of WT or E209A SLFN11 treated with doxycycline (1μg/ml)
531 for 16 hours as visualized by immunoblot analysis. (F) Global translation (x-axis) and caspase-
532 3 cleavage (y-axis) were measured by flow cytometry in polyclonal pools of SLFN11-deficient
533 HAP1 cells transduced with a lentivirus to enable inducible expression of WT or E209A
534 SLFN11. The cells were treated with doxycycline (1μg/ml) for 16 hours, then treated with
536 SLFN11 positivity was gated for in the manner shown in fig. S15A.
537
A SLFN11
negative cells
SLFN11
positive cells
Normalized count
Normalized count
induce SLFN11
w/o w/ doxycycline
doxycycline for 16h
1 2 3 4 5 1 2 3 4 5
10 10 10 10 10 10 10 10 10 10
SLFN11 SLFN11
B
Highly replicating cells
Untreated Etoposide Etoposide+ATRi

5
10 37.8% 3.8% 35.7%
-doxycycline
4
10
3
10
SLFN11 WT
2
10
10
5 39% 0.5% 14.8%
+doxycycline
4
10
3
10
EdU incorporation
2
10
50 100 150 200 250 50 100 150 200 250 50 100 150 200 250
10
5 36.4% 4% 34.8%
-doxycycline
4
10
3
10
E209A SLFN11
2
10
10
5 38.9% 0.2% 12.6%
+doxycycline
4
10
3
10
2
10
50 100 150 200 250 50 100 150 200 250 50 100 150 200 250
DAPI (a.u.)
538
539 Fig S15. A SLFN11 mutant lacking tRNA cleavage activity (E209A) retains replication
540 blocking activity in response to DNA damage. (A) Schematic representation of the gating
541 strategy used for SLFN11 positivity in fig. S14E, fig. S15B, and fig. S16B. (B) DNA (x-axis)
542 and EdU incorporation (y-axis) were measured by flow cytometry in polyclonal pools of
543 SLFN11-deficient HAP1 cells supplemented with inducible WT or E209A SLFN11. The cells
544 were treated with or without doxycycline (1μg/ml) for 16 hours, then treated with etoposide
545 (5μM) for 4 hours with or without the ATR inhibitor VE-821 (10μM) followed by puromycin
546 incorporation (2μg/ml) for 10 minutes.
547
A
SLFN11 WT K652D WT K652D
Doxycycline _ _ + +
SLFN11
α-tubulin
B

10
4 1.9% 2.1% 6% 17.6%
WT SLFN11
3
10
2
Cleaved Caspase 3
10
1
10
2 3 4 2 3 4 2 3 4 2 3 4
10 10 10 10 10 10 10 10 10 10 10 10
10
4 1.3% 1.1% 0.8% 0.6%
K652D SLFN11
3
10
2
10
1
10
2 3 4 2 3 4 2 3 4 2 3 4
10 10 10 10 10 10 10 10 10 10 10 10
Translation
548
549 Fig S16. A SLFN11 mutant defective in ssDNA binding (K652D) does not induce
550 inhibition of translation or apoptosis in response to DNA damage. (A) Expression of
551 SLFN11 in SLFN11-deficient HAP1 cells transduced with a lentivirus to enable inducible
552 expression of WT or K652D SLFN11 treated with doxycycline (1μg/ml) for 16 hours as
553 visualized by immunoblot analysis. (B) Global translation (x-axis) and caspase-3 cleavage (y-
554 axis) were measured by flow cytometry in polyclonal pools of SLFN11-deficient HAP1 cells
555 transduced with a lentiviral vector to mediate doxycycline inducible expression of WT or
556 K652D SLFN11. The cells were treated with or without doxycycline (1μg/ml) for 16 hours,
557 then treated with etoposide (5μM) for 4 hours followed by puromycin incorporation (2μg/ml)
558 for 10 minutes. SLFN11 positivity was gated for in the manner shown in fig. S15A.
559
A sgRNA in exon 14
enrichment for low in ZAKα

ZAKα
ZAKβ
10kb
High
Cleaved
Caspase 3
Low
High
p-JNK
Etoposide
Low
High
p-JNK
Anisomycin
Low
B C
WT ∆SLFN11 ∆ZAKα
Untreated Etoposide
CC3+ CC3+ Cisplatin _ + _ + _ +
2.2% 22.9%
SLFN11
ZAKα
WT
p-EIF2α
Normalized count
2.2% 1.3%
∆SLFN11
Translation
(Puromycin)
0.7% 4.35%
∆ZAKα
p-JNK
α-tubulin
1 2 3 4 1 2 3 4
10 10 10 10 10 10 10 10
Translation
560
561 Fig S17. ZAKa is required for activation of JNK and caspase-3 in response to etoposide
562 and cisplatin. (A) Gene-trap insertion plot of several gene loci in the etoposide treated
563 translation screen. Unique gene-trap insertions (mutations) mapped to several loci in the low
564 or high FACS-sorted channel are shown for the screen. Insertions in the gene body between
565 the start and stop codon in the sense orientation were counted for the mutational index (MI).
566 (B) Example of flow cytometry analysis quantified in Fig. 4h. (C) Parental HAP1, SLFN11-
567 deficient and ZAKa-deficient cells were treated with cisplatin (2.5μM) for 16 hours followed
568 by puromycin incorporation (2μg/ml) for 10 minutes and subjected to immunoblot analysis.
569
A B C
HAP1 A549
_ _ Low translation Leucine
Etoposide + + UAA 3-1
2.1%
SLFN11 1.5
*
Untreated
Relative tRNA level

Normalized count
1.0
Puromycin 41.9%
Etoposide
0.5
0.0
Etoposide - +
1 2 3 4
α-tubulin 10 10 10 10
Translation
D E
2 Etoposide vs untreated UUA

in A549 cells

5
Untreated
(1481 genes)
abundance shift
Treated
Subsequence
1 3
0 0
A R N D C Q E G H I L KM F P S T WY V
Codons
F H I
∆SLFN11
WT
pool Low translation & CC3 positive Low translation & TUNEL positive
SLFN11 (short) Untreated Etoposide Untreated Etoposide
SLFN11 (long)
0.01% 11% 0.05% 13.4%
3 3
α-tubulin 10 10
WT
WT
G 2 2
Cleaved caspase 3
10 10
∆SLFN11
WT
pool
TUNEL
1 1
10 10
_ + _ +
Etoposide
ZAKα 0.06% 0.9% 0.05% 2.2%
P-ZAKα
∆SLFN11
∆SLFN11
3 3
10 10
(long)
2 2
10 10
α-tubulin 1 1
10 10 2 3 4 5
2 3 4 2 3 4 2 3 4 5
10 10 10 10 10 10 10 10 10 10 10 10 10 10
570
571 Fig S18. A549 cells undergo DNA damage-induced apoptosis and ribosomal UUA stalling
572 through SLFN11. (A) HAP1 and A549 cells were treated with etoposide (5μM) for 4 hours
573 (HAP1) or etoposide (25μM) for 12 hours (A549) followed by puromycin incorporation
574 (2μg/ml) for 10 minutes and were subjected to immunoblot analysis. (B) Global translation
575 was measured by flow cytometry in A549 cells treated with etoposide (25μM) for 18 hours
576 followed by puromycin incorporation (2μg/ml) for 10 minutes. (C) Quantitative real time PCR
577 analysis of leucine tRNA-UAA 3-1, normalized to U6 snRNA, in A549 cells treated with
578 etoposide (25μM) for 18 hours. (D) Diricore analysis bar plots depicting differential codon
579 occupation (at position -15 of the RPFs) in etoposide treated (25 μM for 24 hours) versus
580 untreated A549 cells. (E) Metagene density profiles depicting global shifts of RPFs to the start
581 of the coding sequence comparing etoposide treated and untreated A549 cells. The y-axis is
582 intra-gene normalized RPF density. (F) Immunoblot analysis visualizing SLFN11 abundance
583 in polyclonal pools of A549 cells that were transduced with Cas9 and sg1-SLFN11. (G)
584 Phosphorylation of ZAKa determined by Phos-tag gel electrophoresis followed by
585 immunoblot analysis in WT and SLFN11-deficient cells from fig. S18F treated with etoposide
586 (25μM) for 12 hours. (H) Global translation (x-axis) and caspase-3 cleavage (y-axis) were
587 measured by flow cytometry in WT and SLFN11-deficient cells from fig. S18F treated with
588 etoposide (25μM) for 24 hours. (I) Global translation (x-axis) and TUNEL positivity (y-axis)
589 were measured by flow cytometry in WT and SLFN11-deficient cells from fig. S18F treated
590 with etoposide (25μM) for 24 hours.
591
A B C
HAP1 PC3
Leucine
_ _ Low translation
Etoposide + + UAA 3-1
2.9%
1.5
SLFN11
*
Untreated
Relative tRNA level

Normalized count
1.0
50.4%
Puromycin
Etoposide
0.5
0.0
Etoposide - +
2 3 4 5
10 10 10 10
α-tubulin
Translation
D E
Etoposide vs untreated
in PC3 cells
20

(647 genes) UUA 7
Untreated
abundance shift
Subsequence
6 Treated
5
10
4
2
0 1
0
A R N D C Q E G H I L KM F P S T WY V
Codons
F H I
Low translation & CC3 positive Low translation & TUNEL positive
∆SLFN11
WT
pool Untreated Etoposide Untreated Etoposide
SLFN11 (short) 5
10
4 0.04% 16.9%
10 0.19% 12.1%
SLFN11 (long) 4
10
WT
WT
3
10
α-tubulin 3
Cleaved caspase 3
10
2
10
G 10
2
TUNEL
1
10
∆SLFN11 5
WT 10
pool 4
10
0.24% 1.9%
0.09% 2.7%
4
∆SLFN11
∆SLFN11
10
_ + _ +
Etoposide 3
10
3
ZAKα 2
10
10
P-ZAKα 10
2
1
10
2 3 4 2 3 4 2 3 4 2 3 4
10 10 10 10 10 10 10 10 10 10 10 10
α-tubulin
592
593 Fig S19. PC3 cells undergo DNA damage-induced apoptosis and ribosomal UUA stalling
594 through SLFN11. (A) HAP1 and PC3 cells were treated with etoposide (5μM) for 4 hours
595 (HAP1) or etoposide (25μM) for 12 hours (PC3) followed by puromycin incorporation
596 (2μg/ml) for 10 minutes and were subjected to immunoblot analysis. (B) Global translation
597 was measured by flow cytometry in PC3 cells treated with etoposide (25μM) for 12 hours
598 followed by puromycin incorporation (2μg/ml) for 10 minutes. (C) Quantitative real time PCR
599 analysis of leucine tRNA-UAA 3-1, normalized to U6 snRNA, in PC3 cells treated with
600 etoposide (25μM) for 16 hours. (D) Diricore analysis bar plots depicting differential codon
601 occupation (at position -15 of the RPFs) in etoposide treated (25 μM for 18 hours) versus
602 untreated PC3 cells. The cutoff for genes being counted was >25 reads. (E) Metagene density
603 profiles depicting global shifts of RPFs to the start of the coding sequence comparing etoposide
604 treated and untreated PC3 cells. The y-axis is intra-gene normalized RPF density. (F)
605 Immunoblot analysis visualizing SLFN11 abundance in polyclonal pools of PC3 cells that were
606 transduced with Cas9 and sg1-SLFN11. (G) Phosphorylation of ZAKa determined by Phos-
607 tag gel electrophoresis followed by immunoblot in WT and SLFN11-deficient cells from fig.
608 S19F treated with etoposide (25μM) for 24 hours. (H) Global translation (x-axis) and caspase-
609 3 cleavage (y-axis) were measured by flow cytometry in WT and SLFN11-deficient cells from
610 fig. S19F treated with etoposide (25μM) for 48 hours. (I) Global translation (x-axis) and
611 TUNEL positivity (y-axis) were measured by flow cytometry in WT and SLFN11-deficient
612 cells from fig. S19F treated with etoposide (25μM) for 48 hours.
613
A p6T p19bT p26T
C
1.5
_ + _ + _ + in OPTIC14 UUA
Etoposide Etoposide
(1734 genes)
abundance shift
Subsequence
ZAKα ZAKα 1
P-ZAKα P-ZAKα
0.5
α-tubulin α-tubulin
***
A R N D C Q E G H I L K M F P S T W Y V
B ** D Codons
50
1.5
40 in RAS11
(2512 genes)
abundance shift
30
Subsequence
1
20
10 0.5
*
5
ns 0
0
A R N D C Q E G H I L K M F P S T W Y V
T
bT
14
6T
S1
P6
Codons
C
P2
9
A
p1
I
PT
R
O
E F
Patient 6T Patient 19bT
CUA CUC CUG CUA CUC CUG
3 3 3
0.3 0.3 0.3
2 2 2
1 1 1
0 0 0 0 0 0
1 1 1

2 2 2
3 3 3
-30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60
Position in respect to codon Position in respect to codon Position in respect to codon Position in respect to codon Position in respect to codon Position in respect to codon
CUU UUA UUG CUU UUA UUG

3 3 3
0.3 0.3 0.3
2 2 2
1 1 1
0 0 0 0 0 0
1 1 1
2 2 2
3 3 3
-30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60
G H
Patient OPTIC14 Patient 26T
0.6 0.6 0.6 0.3 0.3 0.3
0 0 0 0 0 0

-30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60

0.6 0.6 0.6 0.3 0.3 0.3
0 0 0 0 0 0
-30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60
I J
Patient RAS11 Human T-cells

0.3 0.3 0.3 1 1 1
0 0 0 0 0 0

-30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60

0.3 0.3 0.3 1 1 1
0 0 0 0 0 0
-30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60 -30 -15 0 60
615
616 Fig S20. SLFN11 positive colorectal cancer organoids undergo UUA stalling and activate
617 the ribotoxic stress response in response to DNA damage. (A) Colon cancer organoids p6T,
618 p19bT, and p26T were treated with etoposide (33μM) for 18 hours (p6T and p19bT) or 24
619 hours (p26T). Phosphorylation of ZAKa was determined by Phos-tag gel electrophoresis
620 followed by immunoblot analysis. (B) Bar graph depicting SLFN11 expression per organoid
621 as determined by read counts in the ribosome profiling experiments. P-values were calculated
622 using a two-tailed. t-test, * p<0.05, ** p<0.005, ***p<0.0005. Data are shown as mean ± SD.
623 (C and D) Diricore analysis bar plots depicting differential codon occupation (at position -15
624 of the RPFs) in etoposide treated (33 μM for 22 hours) versus untreated OPTIC0014 and
625 RAS11 organoids. (E to I) Diricore analysis line plots depicting differential
626 (etoposide/untreated) ribosome occupancy (5’-RPF) at -30 to +60 of the leucine codons in
627 control versus etoposide (33 μM) treated organoids: p6T (18 hours), p19bT (18 hours),
628 OPTIC0014 (22 hours), p26T (24 hours), and RAS11 (22 hours). (I) Diricore analysis line plots
629 depicting differential (etoposide/untreated) ribosome occupancy (5’-RPF) at -30 to +60 of the
630 leucine codons in control versus etoposide (10 μM for 12 hours) treated healthy human T cells.
631
A B
Leucine
Untreated Etoposide UAA 3-1
5 1.5
10
Cleaved Caspase 3
*
2.8% 20.8%
Relative tRNA level

4
10
1.0
3
10
2
10 0.5
1
10
0.0
1 2 3 4 1 2 3 4
10 10 10 10 10 10 10 10 - +
Translation
632
633 Fig S21. T-cells undergo inhibition of translation, apoptosis, and tRNA-UAA 3-1 cleavage
634 in response to DNA damage. (A) Global translation (x-axis) and caspase-3 cleavage (y-axis)
635 were measured by flow cytometry in healthy primary human T-cells treated with etoposide
636 (10μM) for 12 hours. (B) Quantitative real time PCR analysis of leucine tRNA-UAA 3-1,
637 normalized to U6 snRNA, in healthy primary human T-cells treated with etoposide (10μM) for
638 12 hours.
639
640 Table S1. Mutation frequencies per gene in cell populations exhibiting high and low
641 puromycin signal in untreated cells. High and low reads were aligned to the reference
642 genome (GRCh38), allowing one mismatch. The sense insertions in the high and low
643 populations were compared using a two-sided Fisher’s exact test to determine the significant
644 differences (FDR corrected p<0.05).
645
647 puromycin signal after treatment with etoposide. High and low reads were aligned to the
648 reference genome (GRCh38), allowing one mismatch. The sense insertions in the high and
649 low populations were compared using a two-sided Fisher’s exact test to determine the
650 significant differences (FDR corrected p<0.05).
651
653 cleaved caspase-3 signal after treatment with etoposide. High and low reads were aligned
654 to the reference genome (GRCh38), allowing one mismatch. The sense insertions in the high
655 and low populations were compared using a two-sided Fisher’s exact test to determine the
657
658 Table S4. Mutation frequencies per gene in cell populations exhibiting high and low p-
659 JNK signal after treatment with etoposide. High and low reads were aligned to the
663
664 Table S5. Mutation frequencies per gene in cell populations exhibiting high and low p-
665 JNK signal after treatment with anisomycin High and low reads were aligned to the
669
670 Table S6. TP53 mutation status for each colorectal cancer organoid
671
Organoid P53 mutation SLFN11
status expression
P6T (46) R175H Positive
P19bT (46) R273C Positive
P26T (46) C176R Negative
RAS11 (CRC metastasis from liver) WT Negative
OPTIC0014 (CRC metastasis from liver) R282W Positive
672
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Supplementary Materials for

DNA damage induces p53-independent apoptosis through ribosome stalling

Nicolaas J. Boon et al.

Corresponding authors: Reuven Agami, r.agami@nki.nl; Thijn R. Brummelkamp, t.brummelkamp@nki.nl

Science 384, 785 (2024)

The PDF file includes:

Materials and Methods

Other Supplementary Material for this manuscript includes the following:

C D 2μM etoposide in HAP1

0 50 100 150 200 250 0 50 100 150 200 250

0 50 100 150 200 250 0 50 100 150 200 250

Cell size (FSC-A - a.u.)

Untreated Gleevec Etoposide Gleevec + etoposide

UNT 1:1 CHX Normalized count UNT 1:1 CHX

UNT 10:1 CHX UNT 10:1 CHX

63.9% 36.1% 90.6% 10.4%

UNT 1:10 CHX UNT 1:10 CHX

Relative read density

Average transcript RPF density

Relative tRNA level

gene rank (10 )

SLFN14 SLFN12 SLFN5 SLFN11

α-tubulin α-tubulin α-tubulin

Tyrosine Leucine Leucine Leucine Leucine Leucine

Relative tRNA level

Relative tRNA level

Relative tRNA level

0.5 0.5 0.5 0.5

SLFN11 Untreated Etoposide Untreated Etoposide

Untreated Etoposide Untreated Etoposide

+ Z-VAD-FMK Untreated Etoposide 5ng/ul IFNy 20ng/ul IFNy

Puromycin Cleaved Caspase 3

Untreated Etoposide ATMi Etoposide + ATMi

1 2 3 4 5 1 2 3 4 5 101 102 103 104 105 1 2 3 4 5

1 2 3 4 5 1 2 3 4 5 101 102 103 104 105 1 2 3 4 5

C D Low translation & CC3 positive

Untreated Etoposide 24h Etoposide & ATRi 24h

WT HAP1 etoposide & ATRi 1.3% 19.5% 43.5%

C Low translation & CC3 positive

Untreated Etoposide Untreated Etoposide

Untreated Etoposide Etoposide+ATRi

Untreated Etoposide Untreated Etoposide

enrichment for low in ZAKα

Relative tRNA level

2 Etoposide vs untreated UUA

Average transcript RPF density

Relative tRNA level

Average transcript RPF density

Relative read density

CUU UUA UUG CUU UUA UUG

Relative read density

CUU UUA UUG CUU UUA UUG

CUA CUC CUG CUA CUC CUG

Relative read density

CUU UUA UUG CUU UUA UUG

Relative tRNA level

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