Adipogenesis increases in calreticulin-deficient EBs. Oil red O is a lysochrome used for staining triglycerides and some lipoproteins, thus being useful in visualizing the degree of adipogenesis. (A) At D20, calreticulin-deficient ES cells (G45crt^−/− and L7crt^−/−) stained more extensively with oil red O than WT cells (P < 0.01; n = 6) and made more adipocyte colonies (P < 0.01; n = 5) (B). (C) The ES cells lacking calreticulin (G45crt^−/− and L7crt^−/−) showed an increase in expression of PPARγ2, C/EBPα (by Western blotting), and aP2 (by PCR) compared with the WT or L7 cells. Relative protein levels were determined by normalizing the calreticulin, PPARγ2, and C/EBPα to GAPDH, and aP2 mRNA levels were normalized with the housekeeping gene L32. (D) Quantitative analysis of the relative expression of adipogenic markers at D20 (P < 0.01; n = 6). (E) Oil red O staining and Western blot analysis with anticalreticulin (CRT) antibodies of 3T3-L1 preadipocytes and 3T3-L1 cells overexpressing calreticulin (3T3-L1 + CRT). 3T3-L1 cell fibroblasts overexpressing calreticulin showed reduced oil red O staining. (F) RT-PCR showing that adipogenic marker expression decreased in 3T3-L1 + CRT cells overexpressing calreticulin compared with control 3T3-L1 cells. Error bars represent SD.