Changes in intracellular Ca²⁺ concentration affect adipogenesis. BAPTA-AM, a membrane-permeable Ca²⁺ chelator, was used to lower cytosolic Ca²⁺ concentration, whereas a Ca²⁺ ionophore, ionomycin, was used to increase it. (A) WT and L7 ES cells showed increased oil red O staining after treatment with BAPTA-AM, whereas no substantial change in the staining was discernible in calreticulin-deficient ES cells (G45crt^−/− and L7crt^−/−). Ionomycin treatment decreased oil red O staining in calreticulin-deficient cells. (B) Colorimetric assay of oil red O staining in the ES cells treated as in A (n = 6). (C) Treatment with BAPTA-AM increased the expression of PPARγ2, C/EBPα, and aP2 in WT or L7 ES cell lines. Conversely, ionomycin treatment decreased the adipogenic marker expression in calreticulin-deficient (G45crt^−/− and L7crt^−/−) cells. (D) Quantitative analysis of relative adipogenic marker expression after BAPTA-AM treatment (P < 0.01; n = 6). (E) Quantitative analysis of relative adipogenic marker expression after ionomycin treatment (P < 0.01; n = 6). Error bars represent SD.