Fig. 2

Silencing of STAT3 reduced PDK1 expression. a Activation of STAT3 in melanoma cell lines. Western blot analysis of STAT3 and P-STAT3 levels in human melanoma cell lines. b STAT3 inhibition by SKI-606 reduced PDK1 expression. WM9 cells were treated with 1 uM SKI-606 (SKI) for 8 h. Protein extracts were blotted with the indicated antibodies. PDK1 levels were normalized to the loading control and expressed as the fold change (FC, numbers below the blots) relative to DMSO-treated cells. c, d Silencing of STAT3 in WM9 (c) and UACC903 (d) cells reduced PDK1 expression. Cells were transduced with retrovirus encoding two STAT3 shRNA (I and II) and a scramble sequence as a control. Protein extracts were probed with STAT3, PDK1 and GAPDH or Actin as a loading control. The blots displayed are representative of three independent experiments. Bar graphs show the mean ± S.D. (from three independent experiments) of PDK1 levels normalized to the loading control and expressed as the fold change (FC) relative to scramble cells. Statistical significance was tested by one-tailed Student’s T-Test using log transformed FC values. *p < 0.01, n = 3. e Flow cytometry analysis of PDK1 expression. Cells were collected and stained as described in “Methods”. Light-grey filled histogram correspond to control (preimmune) Ab and open histograms to PDK1 ab. The MFI (Mean Fluorescence Intensity) was detected by flow cytometry. Bar graph shows the mean PDK1 MFI ± S.D. (from three independent experiments). Statistical significance was tested by a one-tailed Student’s T-Test. ***p < 0.0001, n = 3. The blots and histogram displayed are representative of three independent experiments