STAT3 activation by E6 is essential for the differentiation-dependent HPV18 life cycle
Fig 2
HPV E6 promotes the dual phosphorylation and activation of STAT3.
A) Representative western blot of C33a cells transfected with GFP tagged HPV18 oncoproteins E5, E6 or E7 and analysed for STAT3 activation using specific antibodies detecting phosphorylated and total STAT3. Expression of HPV oncoproteins was confirmed using a GFP antibody. GAPDH served as a loading control. B) Representative luciferase reporter assays from C33a cells co-transfected with GFP tagged HPV18 oncoproteins E5, E6 or E7 and either a β-casein promoter reporter plasmid or a PomC reporter plasmid, which contain STAT3 binding sites, and promoter activity measured using a dual-luciferase system. Data are presented as relative to the GFP transfected control cells. Bars represent the means ± standard deviation from at least three independent biological repeats. **P<0.01, N.S. = not significant (Student’s t-test). C) Representative luciferase reporter assays from C33a cells co-transfected with GFP tagged E6 and a β-casein promoter reporter plasmid treated with the STAT3 inhibitors cryptotanshinone and S3I-201, and the STAT5 inhibitor pimozide. Promoter activity was measured using a dual-luciferase system. Data are presented as relative to the GFP transfected control. Bars represent the means ± standard deviation from at least three independent biological repeats. **P<0.01 (Student’s t-test). D) Representative western blot of C33a cells transfected with GFP or GFP tagged HPV18 E6, untreated or treated with the STAT3 inhibitors as above and analysed for total and phosphorylated STAT3 (Y705 and S727), the expression of cyclin D1 and Bcl-xL and GFP to demonstrate expression of the GFP-18E6 fusion protein. GAPDH expression was used as a loading control. Data shown are representative of at least three biological repeats. E-H) C33a cells were transiently transfected with GFP or GFP tagged HPV18 E6 and left untreated or treated with the STAT3 inhibitors as above and RNA was extracted for qRT-PCR analysis of the indicated STAT3 dependent genes. Samples were normalized against U6 mRNA levels. Representative data are presented relative to the GFP transfected control. Bars are the means ± standard deviation from at least three biological repeats. *P<0.05, **P<0.01, ***P<0.001 (Student’s t-test).