Figure 4

Involvement of FOXO1 in induction of p27 ^Kip1 mediated by resveratrol and MG132. A, K562 cells were treated with 5 μM resveratrol or 5 μM MG132 alone or combination for 8 h and real-time RT-PCR was performed. The results are presented as mean of three independent experiments performed in triplicate, and error bars represent standard deviation. B, K562 cells were pretreated with vehicle or actinomycin D for 1 h, then treated with resveratrol alone or combination with MG132 for additional 8 h, then real-time RT-PCR was performed. The results are presented as mean of three independent experiments performed in triplicate, and error bars represent standard deviation. C, K562 cells were treated with 5 μM resveratrol or 5 μM MG132 alone or combination, FOXO1 mRNA and protein levels were investigated using real-time RT-PCR and Western blot, respectively. The results of real-time RT-PCR are presented as mean of three independent experiments performed in triplicate, and error bars represent standard deviation. Representative blot from three independent experiments with similar results was shown. D, K562 cells were treated as C, Western blot was performed on cytosolic and nuclear proteins, respectively. Representative blot from three independent experiments with similar results was shown. E, K562 cells were exposed to MG132 or resveratrol alone or combination for 8 h. Cross-linked chromatin was extracted and immunoprecipitated with an anti-FOXO1 antibody. Immunoprecipitated DNA was amplified by real-time RCR. The results are presented as mean of three independent experiments performed in triplicate, and error bars represent standard deviation. F, K562 cells were transfected with scramble or siRNA against FOXO1 (siFOXO1) for 48 h, then treated with MG132 or resveratrol alone or combination for additional 24 h, and Western blot was performed. Representative blot from three independent experiments with similar results was shown. *, P < 0.01 vs control; #, P < 0.01.