Figure 2.

Calreticulin/Ca²⁺, calcineurin, and NFAT regulates osteoblast and chondrocyte specification. Differentiating WT control, crt^−/−, and crt^−/−+CaN* ESC cultures were treated on days 3–5 with cyclosporin A, BAPTA-AM, and ionomycin or on days 6–17 with A-285222, and then Ca²⁺-based mineralization was measured on day 21 by an Arsenazo III assay (A) or von Kossa (D and E). Acidic proteoglycans were stained with Alcian blue where indicated (D). B and C, RT-qPCR analysis of osteoblast (B) and chondrocyte (C) markers using RNA extracted at the indicated time points during the osteogenic differentiation protocol. WT (■) ESCs were treated with BAPTA/AM (▵), and crt^−/− (♦) ESCs were treated with ionomycin (♢) between days 3 and 5. Data are expressed as means ± S.D. (error bars), and n ≥ 3. A, one-way ANOVA result: p < 0.0001, F = 18.29; B and C, two-way ANOVA: transcript levels in WT control ESCs treated or not with BAPTA-AM (Runx2, p = 0.0002, F = 17.85; osterix, p = 0.0054, F = 8.815; osteocalcin, p = 0.0003, F = 16.21; Sox9, p = 0.5169, F = 0.4296; aggrecan, p = 0.0173, F = 6.261); transcript levels in crt^−/− ESCs treated or not with ionomycin (Runx2, p = 0.0462, F = 4.257; osterix, p = 0.0079, F = 7.931; osteocalcin, p = 0.0004, F = 15.07; Sox9, p = 0.0001, F = 18.65; aggrecan, p = 0.0003, F = 16). Bonferroni post hoc analysis was as indicated (A–C): *, p < 0.05; **, p < 0.01; ***, p < 0.001.