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Fig. 6 | Cell & Bioscience

Fig. 6

From: STAT3 enhances the constitutive activity of AGC kinases in melanoma by transactivating PDK1

Fig. 6

PDK1 mediates STAT3’s effect on melanoma cell survival. a Reconstitution of PDK1 upon STAT3 silencing rescued Akt phosphorylation. WM9-scramble and WM9-shSTAT3 cells were stably transduced with HA-PDK1. Protein extracts were probed with STAT3, P-Akt, Akt, HA and GAPDH 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 P-Akt levels normalized to total Akt and expressed as the fold change relative to scramble/pBH cells (Control). Statistical significance was tested by one-tailed Student’s T-Test using log transformed FC values. **p < 0.001, ns: not significant, n = 5. be PDK1 reconstitution prevents dacarbazine-induced cell death in STAT3 knockdown cells. The cells were treated with dacarbazine (100 μg/ml) for 72 h. b Viability was determined by the Trypan Blue exclusion assay. Bar Graph shows the mean ± S.D. (from three independent experiments) of the percentage of non-viable cells. Statistical significance was tested by ANOVA followed by Tukey’s Multiple Comparison Test. *p < 0.01 **p < 0.001, ns: not significant, n = 3. c Viability was determined by the Resazurin assay. Bar Graph shows the mean ± S.D. (from three independent experiments) of the percentage of non-viable cells. Statistical significance was tested by ANOVA followed by Tukey’s Multiple Comparison Test. *p < 0.01 **p < 0.001, ns: not significant, n = 3. d, e WM9 (d) or UACC903 (e) cells were treated with dacarbazine, stained with PI/Annexin V and analyzed by Flow cytometry. Representative histograms are shown. The ratio of dead/live cells from three biological replicates ± S.D is shown below. Live cells correspond to cells in the lower left quadrant and dead cells are the sum of the other three quadrants. Statistical significance was tested by ANOVA followed by Tukey’s Multiple Comparison Test. *p < 0.01 **p < 0.001, ns: not significant, n = 3. f PDK1 mediates STAT3’s effect on expression of anti-apoptotic proteins. Protein extracts from WM9-scramble and WM9-shSTAT3 cells stably transduced with PDK1 were blotted with the indicated antibodies. Tubulin was used as loading control. Protein levels were normalized to the corresponding loading control and expressed as the fold change (numbers below the blots) relative to scramble cells

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