Fig. 2

The effect of STAT3 knockdown on viability, proliferation, and treatment responses of melanoma cells. a MTT metabolism test and BrdU incorporation test were used to evaluate viability and proliferation of melanoma cells, respectively, 72 h after the transfection with siSTAT3 or control siRNA (taken as 100%). The data represent mean ± s.d. from four independent experiments; statistical significance calculated by Student’s t test, p < 0.05 were considered significant. b Representative histograms show distribution and percentages of cells in various phases of the cell cycle determined by flow cytometry. The cells were transfected with siRNAs, and 72 h later cells were collected, stained with propidium iodide, and analyzed by flow cytometry. The percentage of cells in the sub-G1, G1, S, and G2/M populations was determined by CellQuest software. c Evaluation of doxorubicin or UVC effects on STAT3-depleted cells. Percentages of living melanoma cells transfected with siSTAT3 or control siRNA and treated 48 h later with 5 µM doxorubicin or irradiated with UVC light are presented. MTT metabolism test was performed 24 h after treatment. The results are related to values obtained for the cells transfected with siCtr. The data represent means ± s.d. from four independent experiments; statistical analysis was done by Student’s t test, p < 0.05 are considered significant. Lower panel shows the analysis of cell proliferation in the same cultures. BrDU incorporation assay was performed 24 h after treatment. d Immunoblots show the levels of STAT3, the cell cycle-regulatory protein—CYCLIN D1, and the levels of cleaved PARP in control and STAT3-depleted cells after treatments in two melanoma cell lines. Total protein extracts were collected 24 h post treatment, whole-cell lysates (30 μg) were subjected to SDS-PAGE, blotted, and probed with various antibodies; detection of β-actin was used as a protein loading control