Figure 3.

Effect of GRP knockdown and RC-3095 treatment on Pi-induced apoptosis and matrix vesicle release in VSMCs. A7r5 cells transfected with GRP siRNA or negative control siRNA for 24 h and cultured in calcification medium (2.6 mM) for 5 days. Primary VSMCs were cultured in calcification medium with or without RC-3095 (1 μM) for 5 days. (a) Induction of apoptosis was detected by flow cytometry with PI staining. (b) Apoptosis in A7r5 cells (green) was determined by TUNEL staining. Nuclei were stained with DAPI (blue) (original magnification, ×400). Scale bar: 50 μm. (c) Western blots were individually probed with antibodies against total/cleaved-caspase-3, total/cleaved-caspase-9, Bcl2, Bad and β-actin. (d and e) Matrix vesicles were isolated as described in the Section 2. ALP activity was measured and normalized to the total matrix vesicle protein content. *p < 0.01 vs. control siRNA or vehicle. Data shown are the mean ± SD, obtained from at least three independent experiments.