Calreticulin, PPARγ2, and C/EBPα levels increase over the 20-d differentiation time line, whereas overexpression of calreticulin inhibits adipogenesis. (A–C) Calreticulin (CRT), PPARγ2, and C/EBPα expression was examined in WT and G45crt^−/− cells by Western blotting over 20 d of adipocyte differentiation as described in Materials and methods. The arrows represent the addition of retinoic acid (RA) or insulin (Ins) and TIT (triiodothyronine). (A) 10⁻⁷ M RA treatment of cells during the first 3 d of differentiation induced calreticulin expression in WT cells. (B and C) Expression of PPARγ2 and C/EBPα in RA-treated crt^−/− cells gradually increased, whereas their levels in WT cells remained low over the 20-d differentiation. Standard error bars in A–C are smaller than the symbols. RA treatment up-regulates calreticulin in 3T3-L1 cells. (D) Western blot analysis with anti-HA antibodies of 3T3-L1 preadipocytes (3T3-L1 control) and 3T3-L1 cells overexpressing HA-tagged calreticulin (3T3-L1 + CRT) show a marked increase in calreticulin levels after RA treatment. (E) 3T3-L1 cells treated with RA show a decrease in oil red O staining, whereas overexpression of calreticulin reduced the staining further. Error bars represent SD.