FIGURE 1.

Inhibition of adipocyte differentiation in 3T3-L1 cells by knockdown of p21. A, changes of p21 protein in differentiating 3T3-L1 cells. Total cellular proteins were extracted from cells at the indicated days of differentiation and subjected to immunoblot analysis with antibodies against p21 and α-tubulin as a loading control. B, efficacy of p21 knockdown in 3T3-L1 preadipocytes infected with p21RNAi adenoviruses. Expression of p21 and p27 level is shown by immunoblotting 24 h after the infection of p21 RNAi adenovirus (137i or 275i) or LacZi adenovirus (LacZi) as a negative control. Note that 137i exhibits nearly complete suppression with a higher efficiency of p21 knockdown than 275i. C, effect of p21 gene knockdown on adipocytes differentiation. 3T3-L1 adipocytes were infected with adenoviruses at day 3. The Oil Red O staining of cells from day 6 post-differentiation induction is shown. D and E, time course analysis of protein (D) and mRNA (E) expression in 3T3-L1 cells after infection of p21 RNAi adenoviruses at day 3. Cellular total RNA (5 μg) and protein (80 μg) were subjected to SDS-PAGE followed by immunoblot analysis for p21, p27, and C/EBPα (D) and to Northern blot analysis for PPARγ, adipsin, and C/EBPβ (E), respectively.