Calmodulin, CaMKII, and CREB expression is up-regulated in calreticulin-deficient ES cells. (A) The G45crt^−/− and L7crt^−/− cells had increased calmodulin, phosphorylated CaMKII (Thr286), and phosphorylated CREB (pSer133) expression when compared with WT and L7 cells. (B) Quantitative analysis of the expression of calmodulin, CaMKII, and CREB (P < 0.01; n = 6). (C) 10 μM KN-62 and KN-93 reduced oil red O staining in calreticulin-null (G45crt^−/− and L7crt^−/−) cells. Treatment of cells with KN-92, an inactive analogue of KN-93, had no effect on oil red O staining. (D) Colorimetric analysis showed a significant decrease in oil red O staining in calreticulin-null cells upon CaMKII inhibition (P < 0.01; n = 6). (E) Western blot analysis of PPARγ2, C/EBPα, and aP2 after inhibition of CaMKII showed lower expression of adipogenic markers than in the untreated cells. Treatment of cells with KN-92 had no effect on PPARγ2 and C/EBPα expression. (F) Quantitative analysis of the expression of PPARγ2, C/EBPα, and aP2 levels after CaMKII inhibition (P < 0.01; n = 6). Calcineurin activity is higher in WT ES cells than in calreticulin-deficient cells. (G) At D20, calcineurin activity is higher in WT ES cells than either in calreticulin-deficient (G45crt^−/−) cells or undifferentiated ES cells at D0 (n = 4; P < 0.01). (H) Inhibition of calcineurin with cyclosporin A (CsA) increased oil red O staining in 3T3-L1 cells, whereas expression of constitutively active calcineurin decreased oil red O staining. (I) Colorimetric assay of oil red O staining in the 3T3-L1 cells treated as in H (n = 6). 3T3-L1, control; 3T3-L1 + CsA, CsA-treated preadipocytes; 3T3-L1 + CaN, 3T3-L1 cells overexpressing constitutively active calcineurin (CaN). Error bars represent SD.