Figure 7.

Downregulation of CHOP10 mRNA with CHOP10 siRNA allows a better adipocyte differentiation of RNase L^−/−-MEFs. (A) RNase L^−/−-MEFs were transfected with a siCHOP or a siCT, then cells were collected and mRNAs were analyzed, after RT, by q-PCR with specific primers for CHOP10 or EEF1α (Supplementary Table 1). PCR products were analyzed on 1.2% agarose gel. Photographies of the gels are shown. (B) Cells were stained with oil red O to reveal lipids accumulation after 6 days of differentiation. RNase L^−/−-MEFs transfected with siCT (a and c) and RNase L^−/−-MEFs transfected with siCHOP (b and d) were observed at a magnification of × 40. After staining with oil red O, lipids were quantified by spectrophotometric analysis at 540 nm following elution of cell retained oil red O with ethanol. (e) Quantification of lipids in RNase L^−/−-MEFs transfected with siCT or siCHOP, 6 days after induction of differentiation in ADM. The amount of lipids in RNase L^−/−-MEFs transfected with siCT was set at 100%. Error bars refer to the S.D. obtained in four independent experiments conducted in duplicate. ^**P=0.003 RNase L^−/−-MEFs transfected with siCHOP compared with RNase L^−/−-MEFs transfected with siCT. (C) RNase L^−/−-MEFs transfected with siCT or siCHOP were induced to differentiate during 6 days in ADM. Cells were fixed and then incubated with an antibody against perilipin (red; RNase L^−/−-MEFs transfected with siCT (a) and RNase L^−/−-MEFs transfected with siCHOP (b). DNA was stained with DAPI (blue). (D) RNase L^−/−-MEFs transfected with siCT or siCHOP were treated (+) or not (−) 10 min at 37 °C with insulin after 6 days of differentiation. Total cellular extracts were analyzed by western blot with anti-P-Akt (Ser⁴⁷³), anti-Akt and anti-α-tubulin-specific antibodies. (E) Protein bands shown in (D) were quantified with the ImageJ software. Levels of P-Akt (Ser⁴⁷³)/Akt were corrected with corresponding α-tubulin levels, used as indicator of proteins loading. P-Akt (Ser⁴⁷³)/Akt rate in RNase L^−/−-MEFs transfected with siCT or siCHOP before insulin treatment was set at 1. Error bars refer to the S.D. obtained in four independent experiments. NS, non statistically significant P-Akt (Ser⁴⁷³)/Akt level after insulin treatment in differentiated RNase L^−/−-MEFs transfected with siCHOP compared with differentiated RNase L^−/−-MEFs transfected with siCT