Fig 2: ERBB2 controls cervical cancer cell viability and invasion by regulating PIK3CA protein expression. (A) Schematic diagram of the ERBB2-ERRB3 complex interacting with PI3K(p85), thereby promoting the downstream phosphorylation of AKT and mTOR. (B) IP in cervical cancer cell lysates with antibodies against ERBB3 or IgG control. Expression levels of ERBB3, ERBB2 and PI3K(p85) in the IP fraction were assessed via WB. PIK3CA mRNA expression in transfected (C) HeLa and (D) SiHa cells assessed via RT-qPCR. GAPDH was used as the housekeeping control. PIK3CA, p-AKT/AKT and p-mTOR/mTOR protein expression in transfected (E) HeLa and (F) SiHa cells assessed via WB. GAPDH was used as the loading control. (G) Invasion of transfected HeLa cells assessed via Transwell assay. (H) Cellular viability of transfected HeLa cells quantified using Cell Counting Kit-8. (I) Sphere-formation of transfected HeLa cells. Data are expressed as the mean ± SEM (n=3). *P<0.05 and **P<0.01 vs. siCtrl or Ctrl vec; †P<0.05 and ††P<0.01 vs. siERBB2 or ERBB2 vec. Data were analyzed via one-way ANOVA. IP, immunoprecipitation; WB, western blotting; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering; vec, vector; ERBB2, Erb-B2 Receptor Tyrosine Kinase 2; p-, phosphorylated; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α.

Fig 3: In silico analysis of HER2 and HER3 promoter sequences. (a) 1.5 kb promoter region of HER2 gene was fetched from database (ensemble.org) and subjected to transcriptional factor binding prediction program (http://consite.genereg.net/) to predict for putative NRF2 binding sites as indicated. Line with arrowheads shows the 0.7 kb sequence of HER2 promoter [8], while regions enclosed in rectangles show additional sequences included and cloned in the PGL3 luciferase reporter vector (Promega) because of carrying additional NRF2 binding sites. (b) The same analyses were performed for HER3 promoter. In (a) and (b), +1 indicate the transcriptional start site, sequences highlighted in green show NRF2 binding sites as predicted by ConSite, and sequences in bold represent manual identification of putative NRF2 binding sites based on ARE consensus sequence [9] while those highlighted in pink show overlapping NRF2 binding sites by the two methods mentioned above.

Fig 4: HCV induces the expression of the ErbB3 ligands NRG1 and Epiregulin.(A) Total RNA was isolated from Huh9-13 replicon cells or Huh7 control cells. Transcript levels of NRG1, NRG2 and Epiregulin were quantified by rtPCR as outlined in figure legend to Fig 1 and the Material and Methods section using primer pairs described in the supporting information (Table A in S1 File). (B) Supernatants from cell cultures of Huh9-13 cells comprising the subgenomic HCV replicon or Huh7 cells for control were analysed for production and release of soluble NRG1 using ELISA specifically recognizing human NRG1. (C) Huh7.5 cells were infected with the HCVcc strain JC1. Total RNA was prepared 72 hours after infection and NRG1 was determined by rtPCR. For (A) to (C) the results are expressed as fractions of the normalized value of the control which was set to 1; data are presented as mean + SEM of at least three independent experiments.

Fig 5: Reduction of ErbB3 expression on the cell surface is accompanied by increased cell surface expression of EGFR and ErbB2.(A) and (B) Huh9-13 replicon cells were analysed by FACS to determine surface expression of ErbB3 and EGFR (A) or of ErbB3 and ErbB2 (B). (C) and (D) Huh7 cells were transfected with ErbB3 specific siRNA or with control siRNA. 72 hours later cells were prepared for FACS analysis to determine the amounts of ErbB3 and EGFR (C) or of ErbB3 and ErbB2 (D) on the cell surface. Data were analysed using the FlowJo software against the corresponding isotype controls and analysis of EGFR (A and C) or ErbB2 (B and D) surface expression was gated to cells with low expression of ErbB3. Results correspond to the average of the receptors at the cell surface (in precent); error bars indicate SEM from at least 3 independent experiments.