TBHQ treatment causes protein induction of HER2 and HER3 and upregulation of pAKT levels in PEO1 and SKOV3 cells. (a) Immunoblot analysis following treatment with tBHQ demonstrated protein induction of both HER2 and HER3 receptors and increase of pAKT. Exponentially growing cells were either left untreated (UT) or treated with 200 μ
M tBHQ for 4 h before being harvested and processed for immunoblotting using relevant antibodies (Table 1
). (b) Bar chart showing total HER2, total HER3, and phospho-AKT levels in PEO1 and SKOV3 cell lines by quantifying immunoblot signal intensities obtained in (a) and normalised to the value of UT and expressed as fold change. (c) Immunofluorescent labelling of endogenous HER2 and phospho-AKT reveals protein induction following tBHQ treatment. Cells were processed for immunocytochemistry and immunolabelled using anti HER2 (red fluorescence) or phospho-AKT (green fluorescence) primary antibodies followed by Alexa Fluor conjugated secondary antibodies. Nuclear reference was provided by costaining with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI). Scale bar indicates 10 μ
m. (d) Analysis of colocalisation between immunostained HER2 and pAKT in the images obtained in (c). Spatial correlation between the two fluorescent signals was obtained by generating cytofluorograms and performing Pearson’s correlation analysis.