Fig. 5.

Inhibition of STAT3 induces increased expression of nuclear p21. a Cells were pre-incubated with the indicated concentration of stattic and dasatinib, and then treated with 9 μM tamoxifen for 72 h. Cell viability was measured using a CCK-8 kit. b MCF-7 cells and TamR cells were transfected with siControl and siSTAT3 (100 nmol/L). Thereafter, cells were treated with 9 μM tamoxifen for 72 h. Knockdown of STAT3 protein expression was determined using immunoblotting, and then cell viability was measured using a CCK-8 kit. c Fresh whole cell lysates from MCF-7 and TamR were separated into nuclear (Lamin B1) and cytoplasmic (GAPDH) fractions. Expression level of p21 protein was determined using immunoblotting. d After 4 h of pre-incubation with the indicated concentration of stattic, cells were treated with tamoxifen. Fresh whole cell lysates were fractionated and immunoblotted for p21 protein expression. W: whole cell lysates; C: cytoplasmic fraction; N: nuclear fraction. Expression of GAPDH and Lamin B indicates the purity of the cytoplasmic and nuclear fractions, respectively. The cropped blots are used in the figure. The membranes were cut prior to exposure so that only the portion of gel containing the desired bands would be visualized. Each experiment was performed at least three times; representative results are shown. Data are expressed as mean ± SD and all experiments were performed in triplicate, independently. P values calculated using an unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001