Oxidative Medicine and Cellular Longevity / 2016 / Article / Fig 7

Research Article

NRF2 Regulates HER2 and HER3 Signaling Pathway to Modulate Sensitivity to Targeted Immunotherapies

Figure 7

NRF2 knockdown causes downregulation of HER2 and HER3 levels, repression of pAKT, and sensitisation to targeted immunotherapeutics. (a) Immunoblotting analysis showing inhibition of RTK signaling following depletion of NRF2 mRNA by SiRNA in SKOV3 cell line. Exponentially growing cells were either transfected with scrambled SiRNA (Scrmbl) or transfected with 75 pmol of NRF2 SiRNA for either 24 or 48 h or 100 pmol of NRF2 SiRNA for 48 h and processed for immunoblotting using relevant antibodies (Table 1). β-actin was used as a loading control. Bar chart shows protein levels by quantifying immunoblot signal intensities obtained and normalised to the value of untreated (UT) control and expressed as fold change. (b) Immunofluorescent labelling of endogenous total HER2 or phospho-AKT exhibits repression following NRF2 knockdown. Cells were transfected as in (a) and processed for immunocytochemistry. Relevant primary antibodies were used to stain HER2 or phospho-AKT followed by Alexa Fluor conjugated secondary antibody (red fluorescence). Nuclear reference was provided by costaining with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI). Images were captured with Leica DMiRe2 electronic microscope at 100x objective while merging, colocalisation, and further analysis were performed by using integrated features of Andor iQ Core software (ANDOR Technologies Ltd.). Scale bar indicates 10 μm. (c and d) HER2 and HER3 downregulation following NRF2 knockdown is caused by their transcriptional repression. Exponentially growing PEO1 cells (c) or SKOV3 cells (d) were transfected with either empty PGL3 basic vector or 1 μg PGL3 basic vector with cloned 1.5 kb fragments of either HER2 (prHER2) or HER3 (prHER3) upstream promoter regions driving the expression of luciferase gene. Cotransfection with 0.2 μg pRL-CMV plasmid was performed as an internal transfection control. At 24 h after transfection, cells were either left untreated (UT) or treated with 200 μM tBHQ as indicated for 4 h following which, cells were processed for dual luciferase reporter assay (Promega) to record luciferase activity in multiplate reader (MODULUS, Promega). (d) The same was done for SKOV3 cell lines. (e) Knockdown of NRF2 through SiRNA sensitises cancer cell to RTK inhibitors while parallel knockdown of KEAP1 partially relieves this sensitisation. Cells were transfected with scrambled SiRNA or SiRNA targeting NRF2 either alone or with the inclusion of KEAP1 SiRNA. Following further 24 h incubation, cells were either left untreated or treated with 25 μg/mL of HER2 inhibitors Pertuzumab and Trastuzumab. Cytotoxicity assay was performed as in (a). In (c–e), data are the means with ±S.D. of triplicates and expressed as fold change with statistical significance determined by ONE WAY ANOVA followed by Tukey’s post hoc test (for c and d), or Student’s -test (for e) according to the scale :  , :  , and :  .
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