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. 2020 Mar 17;9(3):737. doi: 10.3390/cells9030737

Figure 2.

Figure 2

Figure 2

Effect of GRP silencing and RC-3095 treatment on Pi-induced osteogenic differentiation in VSMCs. A7r5 cells were transfected with GRP siRNA or negative control siRNA for 24 h and cultured in calcification medium (2.6 mM). Primary VSMCs were cultured in calcification medium with or without RC-3095 (1 μM). After 5 days in culture, VSMC calcification was determined by ARS staining (a), calcium content assay (b) and ALP activity assay (c). * p < 0.01 vs. control siRNA or vehicle. (d) Western blots were individually probed with antibodies against Runx2, calponin, p-Smad1/5, Smad1/5, p-ERK1/2, ERK1/2, p-p38MARK, p38MAPK or β-actin. (e) Expression of Runx2 (red) and calponin (green) was examined by fluorescence immunocytochemistry using specific antibodies. Nuclei were stained with DAPI (blue) (original magnification, ×400). Scale bar: 50 μm. (f) Using real time RT-PCR, the expression levels of Runx2 and calponin mRNA were quantified. * p < 0.01vs. control siRNA or vehicle. Data shown are the mean ± SD, obtained from at least three independent experiments.