Figure 5.

Effect of CypB on cell cycle progression during 3T3-L1 cell differentiation. (A) 3T3-L1 cells at 70-80% confluence were transfected with scrambled siRNA (siControl) or CypB siRNA (siCypB). Then the fully confluent cells were differentiated into adipocyte with MDI treatment for 24 h. Cell cycle progression was monitored with flow cytometric analysis. 3T3-L1 cells were stained with propidium iodide nuclear dye under the indicated conditions. Cellular DNA content was analyzed by flow cytometric analysis and cells were distributed in the three phases of the cycle (G₁. S and G₂/M). Results were presented in percentage for each plot and are representative of three independent experiments. (B) The expressions of cell cycle-associated genes (CyclinD, CylclinE, CyclinA, CDK4, p27 and pRB) were analyzed in CypB knockdown by western blotting. Vinculin was used as a loading control. (C) 3T3-L1 cells transfected with pcDNA or CypB-HA were induced to differentiate into adipocytes. Following CypB overexpression, cell cycle progression was monitored as before. (D) Following overexpression of CypB, the expression of cell cycle-associated proteins (CyclinD, CylclinE, CyclinA, CDK4, p27 and pRB) was analyzed by western blotting. The values were shown as the mean ± standard deviation of three independent experiments. CypB, cyclophilin B; si, short interfering; p-phosphorylated.