Fig. 5

iRHOM2 regulates oxidative stress in hyperproliferative keratinocytes. a Quantification of dihydroethidium (DHE) staining in control (CTRL) and TOC keratinocytes by flow cytometry. Data are expressed as mean and SEM of three independent experiments. Statistical analysis was performed to compare CTRL and TOC cells using Student’s two-tailed t-test (**p < 0.01). b DHE staining (red) in live Sh Scr or Sh iRHOM2-transfected CTRL and TOC keratinocytes by confocal microscopy. Hoechst 33342 (blue) was used to identify nuclei. c qRT-PCR of HMOX1, NQO1, SOD and CYGB in TOC keratinocytes was assessed as fold change compared to CTRL cell expression (dashed line). The graph represents means and SEM of three biological replicates. Statistical analysis was performed comparing CTRL and TOC keratinocytes using Student’s two-tailed t-test (*p < 0.05, **p < 0.01, ***p < 0.001). d Immunostaining of CYGB was performed in fore-paw and back skin sections of rhbdf2^+/+ and rhbdf2^−/− mice by confocal microscopy. DAPI (blue) is used as nuclear stain. Scale bar: 20 µm. e Representative WB for CYGB expression from proteins extracted from back skin and fore-paw of rhbdf2^+/+ and rhbdf2^−/− mice. GAPDH was used as a loading control. f WB analysis of CYGB in Sh Scr and Sh iRHOM2-transfected CTRL and TOC keratinocytes. GAPDH was used as a loading control. g Representative confocal images from PLA experiments between iRHOM2 and CYGB in control keratinocytes. PLA between K6 and K16 was performed as a positive (+) control, while no primary antibodies were applied in negative (−) control