Oxidative Medicine and Cellular Longevity / 2016 / Article / Fig 5

Research Article

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

Figure 5

Knockdown of NRF2 by SiRNA causes repression of phospho-NRF2 and HO-1 levels and elevation of reactive oxygen species (ROS). (a) Optimization of SiRNA-mediated NRF2 knockdown. Exponentially growing cells were transfected either with scrambled RNA (scrmbl) or with different amounts of SiRNA for either 24 h or 48 h before being processed for immunoblotting. (b) NRF2 knockdown results in repression of its substrates. The same lysates as in (a) were blotted for phospho-NRF2 and HO-1 levels. Bar charts in (a) and (b) show total NRF2, phospho-NRF2, and HO-1 levels in SKOV3 cell lines by quantifying immunoblot signal intensities obtained in respective blots and normalised to the value of UT and expressed as fold change. (c) NRF2 knockdown leads to ROS accumulation. SKOV3 cells were seeded in triplicate for 18 h and transfected with NRF2 SiRNA. Following 48 h incubation, cells were assayed for total ROS by loading them with DCFDA for 45 min and measuring fluorescence using fluorescence multiplate reader (MODULUS, Promega) with excitation and emission spectra of 485 nm/535 nm. The fluorescence reading was normalised to total cell abundance within the same wells as described in Materials and Methods. Data are the means with ±S.D. of triplicates, normalised to untreated (UT) control and expressed as fold change with statistical significance determined by Student’s -test according to the scale :  , :  , and :  . (d) Immunofluorescent labelling of endogenous phospho-NRF2 and HO-1 exhibits repression following NRF2 knockdown. Cells were transfected as in (a) and processed for immunocytochemistry. Relevant primary antibodies followed by Alexa Fluor conjugated secondary antibodies were used for immunolabelling for phospho-NRF2 and HO-1 (red fluorescence). Nuclear reference was provided by costaining with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI). Images were captured with Leica DMiRe2 electronic microscope with 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.

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