Figure 6

NPAS2 promotes G1 to S phase transition of cell cycle by CDC25A-mediated activation of CDK2/4/6. (a) Cell cycle analysis by flow cytometry in HLE and HLF cells after transiently transfected with siRNA or expression vector as indicated. The percentage of cell population at different cell cycle phases are shown in the histograms and statistically analyzed using a two-tailed t-test. (b) Western blot analyses for NPAS2, CDK2, phosphorylated CDK2 at T14/Y15 (p-CDK2), CDK4, CDK6, phosphorylated CDK6 at Y24 (p-CDK6), cyclin A2, cyclin D1 and cyclin E in HLE and HLF cells treated as indicated. (c) HLE and HLF cells transfected with siRNA or expression vector as indicated. Tyrosine phosphorylation (P-Tyr) was detected by western blot after IP with anti-CDK4 antibodies. (d) Cell cycle analysis by flow cytometry was performed in HLE and HLF cells with treatment as indicated. (e) Western blot analyses for NPAS2, CDC25A, p-CDK2 (T14/Y15), p-CDK6 (Y24) in HLE and HLF cells with treatment as indicated. (f) Tyrosine phosphorylation (P-Tyr) was detected by western blot after IP with anti-CDK4 antibodies in HLE and HLF cells with treatment as indicated. Experiments were repeated twice with independent cell extracts and representative data were presented. Data shown are the mean±S.E.M. from three independent experiments. *P<0.05; **P<0.01