Fig. 4. GZMB counteracted osteoblastic differentiation and calcification of VSMCs under hypoxia.

a Plasmid aimed at overexpression of GZMB was transfected into PASMCs and then exposed to hypoxia for 24 h. Expression of Runx2, MSX2, BMP2, SOX9, and SM22α was evaluated with western blotting. β-Actin served as the standard; n = 8. b Expression of Runx2, MSX2, BMP2, SOX9, and SM22α was evaluated with real-time PCR; 18 s served as the standard; n = 6. c PASMCs were cultured under hypoxia for 7 days upon treatment with procalcifying media. Vascular calcification was assessed by Alizarin Red S staining and determination of calcium content; n = 6. d GZMB plasmid was transfected into PASMCs and exposed to hypoxia for 24 h. Expression of STIM1 and ORAI1 was evaluated with western blotting. β-Actin served as the standard; n = 8. e Overexpression of GZMB was achieved with plasmid and several classical signaling pathways were evaluated with western blotting. β-Actin served as the standard; n = 6. f IWP-2 (27 nM) was administered to inhibit the non-canonical Wnt signals and expression of STIM1 was evaluated with western blotting. β-Actin served as the standard; n = 8. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 versus normoxia group. ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001 versus hypoxia + negative control group. CON, control; ZMB, granzyme B plasmid; HYP, hypoxia; GNC, negative control; NOR, normoxia