Autocrine STAT3 activation in HPV positive cervical cancer through a virus-driven Rac1—NFκB—IL-6 signalling axis
Fig 6
HPV E6 mediated IL-6 expression requires active NFκB.
A) Representative luciferase reporter assay from C33A cells co-transfected with GFP tagged E6 and a ConA reporter containing tandem κB binding sites [93]. Promoter activity was measured using a dual-luciferase system. Data are presented as relative to the GFP transfected control. B) Representative western blot of C33A and normal human keratinocytes (NHK) cells transiently transfected with GFP or GFP tagged HPV18 E6 and analysed for phosphorylated and total p65 expression. Expression of HPV E6 was confirmed using a GFP antibody in the C33A cells. In NHK cells an antibody directly detecting HPV18 E6 was used to confirm expression of the GFP E6 fusion protein. GAPDH served as a loading control. C) Representative western blot of HeLa cells transfected with a pool of two specific siRNAs targeting HPV18 E6 and analysed for the expression of phosphorylated and total p65. Knockdown of HPV18 E6 was confirmed using an antibody against HPV18 E6 and p53. GAPDH served as a loading control. D-F) C33A cells were co-transfected with GFP, GFP tagged HPV18 E6 or GFP tagged HPV18 E6 and mutant IκBα (IκBm). Cells were then either left untreated or treated with IKK inhibitor VII (IKKi). D) Total RNA was extracted for RT-qPCR analysis of IL-6 expression. Samples were normalized against U6 mRNA levels. Representative data are presented relative to the GFP control. E) Cell lysates were analysed for the expression of phosphorylated and total p65 and IL-6. Expression of HPV E6 was confirmed using a GFP antibody and GAPDH served as a loading control. F) The culture medium was analysed for IL-6 protein by ELISA. G-I) HeLa and CaSKi cells were transfected with mutant IκBα (IκBm) or treated with IKK inhibitor VII (IKKi). G) Total RNA was extracted for RT-qPCR analysis of IL-6 expression. Samples were normalized against U6 mRNA levels. Representative data are presented relative to the DMSO or pcDNA control. H) Cell lysates were analysed for the expression of phosphorylated and total p65 and IL-6. GAPDH served as a loading control. I) The culture medium was analysed for IL-6 protein by ELISA. Bars represent the means ± standard deviation from at least three independent biological repeats. *P<0.05, **P<0.01, ***P<0.001 (Student’s t-test).