Fig 2: Schematic demonstrating E7-mediated upregulation of KATP channel expression and activity.HPV E7 upregulates expression of ABCC8, the gene encoding SUR1 which constitutes the regulatory subunit of KATP channels. Increased KATP channel activity contributes towards the activation of MAPK and AP-1 signalling. This, in turn, drives transcription from the viral URR and changes in host gene expression which together stimulate proliferation. Figure created using BioRENDER.com.

Fig 3: KATP channels drive proliferation in cervical cancer cells.A–C Growth curve analysis (A) colony formation assay (to measure anchorage-dependent growth) (B) and soft agar assay (to measure anchorage-independent growth) (C) of HeLa, SiHa and C33A cells after treatment with DMSO or glibenclamide (10 μM) for 24 h. D–F Growth curve analysis (D), colony formation assay (E) and soft agar assay (F) of HeLa and SiHa cells after transfection of SUR1-specific siRNA. Data shown is means ± SD of three biological replicates with individual data points displayed where appropriate. Ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test).

Fig 4: HPV enhances expression of the SUR1 subunit of KATP channels.A mRNA expression levels of KATP channel subunits in HPV- normal human keratinocytes (NHK) and a panel of five cervical cancer cell lines – one HPV- (C33A), two HPV16+ (SiHa and CaSki), and two HPV18+ (SW756 and HeLa) detected by RT-qPCR. Samples were normalised against U6 mRNA levels. Data are displayed relative to NHK controls. B ABCC8 mRNA expression in NHKs and keratinocytes containing episomal HPV18 genomes detected by RT-qPCR. Samples were normalised against U6 mRNA levels. C Representative immunofluorescence analysis of sections from organotypic raft cultures of NHK and HPV18+ keratinocytes detecting SUR1 levels. Nuclei were visualised with DAPI and the white dotted line indicates the basal layer. Two donor cell lines were used to exclude donor-specific effects. Images were acquired with identical exposure times. Scale bar, 40 µm. D ABCC8 mRNA expression in a panel of cervical cytology samples representing normal cervical tissue (N) and cervical disease of increasing severity (CIN 1 - 3) detected by RT-qPCR (n = 5 from each grade). Samples were normalised against U6 mRNA levels. E Representative immunofluorescence analysis of tissue sections from cervical lesions of increasing CIN grade. Sections were stained for SUR1 levels (green) and nuclei were visualised with DAPI (blue). Images were acquired with identical exposure times and the white dotted line indicates the basal layer. Scale bar, 40 µm. F Representative immunofluorescence analysis of sections from organotypic raft cultures of NHK and a W12 cell line presenting with HSIL morphology detecting SUR1 levels (red). Nuclei were visualised with DAPI (blue) and the white dotted line indicates the basal layer. Images were acquired with identical exposure times. Scale bar, 40 µm. G Representative immunohistochemistry analysis and scatter dot plots of quantification of normal cervical (n = 9) and cervical cancer (n = 39) tissue sections stained for SUR1 protein. Scale bar, 50 µm. H Scatter dot plot of expression data acquired from the GSE63514 dataset. Arbitrary values for ABCC8 mRNA expression in normal cervix (n = 24), CIN1 lesions (n = 14), CIN2 lesions (n = 22), CIN3 lesions (n = 40) and cervical cancer (n = 28) samples were plotted. Bars represent means ± SD of three biological replicates, unless stated otherwise, with individual data points displayed where possible. Ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test).

Fig 5: Depletion of SUR1 reduces HPV gene expression in cervical cancer cells.A, B Relative expression of ABCC8 mRNA in (A) HeLa and SiHa cells transfected with a pool of SUR1-specific siRNA (n = 4) and (B) two monoclonal HeLa cell lines stably expressing SUR1-specific shRNAs measured by RT-qPCR. Samples were normalised against U6 mRNA levels. C Relative mean DiBAC4(3) fluorescence levels in HeLa and SiHa cells transfected with SUR1 siRNA. Samples were normalised to scramble controls. D, E Relative expression of E6 and E7 mRNA in (D) HeLa and SiHa cells transfected with SUR1 siRNA (n = 4) and (E) HeLa cell lines stably expressing SUR1-specific shRNAs measured by RT-qPCR. Samples were normalised against U6 mRNA levels. F, G Representative western blots of E6 and E7 expression in (F) HeLa and SiHa cells transfected with SUR1 siRNA and (G) HeLa cell lines stably expressing either a non-targeting (shNTC) or a SUR1-specific shRNA. GAPDH served as a loading control. H Relative firefly luminescence in HeLa and SiHa cells co-transfected with SUR1 siRNA and either a HPV18 or HPV16 URR reporter plasmid respectively (n = 4 for all conditions). Luminescence values were normalised against Renilla luciferase activity and data is displayed relative to scramble controls. Bar graphs represent means ± SD of three biological replicates (unless stated otherwise) with individual data points displayed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test).