Further, the western blot assay revealed that the protein levels of ALP,
Runx2, and OPN were notably up-regulated in a time-dependent manner (P< 0.001, Figure 1D and E).
Runx2 regulates the expression of bone-related proteins such as osteocalcin, osteopontin, as well as receptor activator of nuclear factor kappa-B ligand (RANKL), and alkaline phosphatase (ALP).[8] Osterix is located downstream of
Runx2, and its main role is to increase the expression of osteopontin and ALP.[9] Thus, VSMCs undergo phenotypic transformation and downregulation of expression of the contractile proteins smooth muscle (SM) 22 and alpha-smooth muscle actin (a-SMA).
No statistically significant differences were observed for the expression of the proinflamatory cytokines IL1A, IL6, IL8 and TNF and bone formation genes ALP, COL1 and
RUNX2 in HPDL cells treated with PC 15% or MTA or control cells (Figure 6).
RUNX2 transcription factor mRNA increased by 12-18-fold in differentiated OLC and EXP-21 cells with respect to the undifferentiated hDPSC and remained high at all of the evaluated points, whereas the OSX transcripts reached a significant peak after 7 days of OLC differentiation (200-fold change).
The result showed that, in both arrangements, without use of osteogenic medium, there was an increase in bone related genes
RUNX2 and OCN.
One such example is maternal expression gene 3 (MEG3) regulating the expression of Bmp4,
Runx2, and Osx in human mesenchymal stem cells [12, 13].
The membranes were then incubated with primary antibodies (
Runx2, PPAR[gamma]-2, GSK-3[beta], [beta]-catenin, Dkk-1, and GAPDH, all 1: 1000 dilution) overnight at 4[degrees]C and washed 3 times with TBST for 5 min each before incubation with the secondary antibody at room temperature for 1 h.
After osteogenic differentiation was induced in MSCs cultured for 21 days, the expression of osteogenesis-associated proteins
RUNX2, OPN, and OCN, was analyzed with Western blotting.