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Fig. 5 | Cell & Bioscience

Fig. 5

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

Fig. 5

STAT3 binds to the PDK1 promoter and regulates its transcription. a STAT3 silencing decreased PDK1 mRNA levels. Relative levels of PDK1 mRNA were determined by Real-Time PCR in both UACC903 and WM9 cells stably transduced with two STAT3 shRNA targeting sequences. mRNA levels were normalized to internal RNPII levels and expressed as relative to control cells. For WM9 cells, the mean ± S.D. from three independent experiments is shown. *p < 0.05. For UACC903 cells, the mean ± S.D. from one representative experiment out of two is shown. b Structure of the proximal region of the human PDK1 promoter. Putative STAT3 responsive elements (in grey) and fragments of the promoter that were cloned into pGL2 are depicted. The sites at − 265 and − 137 were removed by mutagenesis. c Silencing of STAT3 diminishes the reporter activity of the PDK1 promoter. The indicated reporter plasmids were transfected into UACC903-shSTAT3 and UACC903-scramble cells. Results are shown as the mean ± S.D. *p < 0.05. **p< 0.01. d Active STAT3 induced luciferase activity driven by the PDK1 promoter. The plasmid − 410/+ 82 was co-transfected into WM115 cells together with STAT3β and STAT3C plasmids. Results are shown as the mean ± SD. **p < 0.01. e STAT3 silencing decrease binding of STAT3 to the PDK1 promoter. The plot shows the relative level of PDK1 amplification (normalized to GAPDH levels) following a Chromatin immunoprecipitation assay on UACC903-shSTAT3 and UACC903-scramble cells. The mean ± S.D. from three independent experiments is shown. *p < 0.05. f STAT3 enhances PDK1 transactivation through the SREs at − 265 and − 137. Reporter plasmids described in b were transfected into UACC903-shSTAT3 and UACC903-scramble cells. Results are shown as the mean ± S.D. *p < 0.001, Student’s t-Test; the activity of the mutant promoters was compared to that of − 410/+ 82 fragment. #p < 0.001, ANOVA followed by Dunnett’s Multiple Comparison Test

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