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. 2018 Apr 9;14(4):e1006975. doi: 10.1371/journal.ppat.1006975

Fig 7. Suppression of STAT3 impairs cell cycle progression and HPV genome maintenance in undifferentiated keratinocytes.

Fig 7

A) Representative western blots from HPV18 containing keratinocytes treated with increasing doses of the STAT3 inhibitor cryptotanshinone and analysed with antibodies specific for cyclin D1 and p21. GAPDH served as loading control. Data are representative of at least three biological independent repeats. B) RNA was extracted from HPV18 containing keratinocytes incubated with 10 μM cryptotanshinone and analysed by qRT-PCR for levels of ccnd1 and p21 mRNA. Samples were normalised against U6 mRNA levels and presented relative to the DMSO control. Bars represent means ± standard deviation of at least three biological repeats. **P<0.01 (Student’s t-test). C) Representative western blots of HPV18 containing keratinocytes transfected with a panel of 4 STAT3 specific siRNA and a scrambled control and analysed by western blotting using antibodies specific for cyclin D1 and p21. GAPDH served as loading control. Data are representative of at least three biological independent repeats. D) Representative western blots from HPV18 containing keratinocytes transduced with a lentivirus encoding a dominant negative Y705F phospho-site mutant or transiently transfected with a dominant negative STAT3 S727A mutant and analysed by blotting with antibodies specific for cyclin D1 and p21. GAPDH served as loading control. Data are representative of at least three biological independent repeats. Representative flow cytometric analysis of the cell cycle in E) HPV18 containing keratinocytes or F) normal human keratinocytes treated with 10 μM cryptotanshinone. G) As in E except cells treated with a pool of four STAT3 specific siRNAs. Data are expressed as percentage of cells at each stage of the cell cycle. Bars represent means ± standard deviation of at least three biological repeats. *P<0.05, ***P<0.001 (Student’s t-test) compared to DMSO or scrambled control cells. H) Representative Southern blot analysis of undifferentiated HPV18 containing keratinocytes treated with 10 μM cryptotanshinone or a pool of four STAT3 specific siRNAs.