Fig. 4: Caspase-8 phosphorylation on Tyrosine 380 sustains NRF2 activation.
A Immunofluorescence in U87shCTR, U87shC8#2, U87shC8#2 + C8WT and U87shC8#2 + C8Y380F cells: NRF2 (green) and DNA (Hoechst, blue). B Quantification of NRF2 staining in A) reported as the ratio between nuclear and cytosolic fluorescence intensity (N/C). C Immunoblotting and relative densitometric analyses of GR, HO-1 and NQO1 in U87shCTR, U87shC8#2, U87shC8#2 + C8WT and U87shC8#2 + C8Y380F cells. Vinculin was used as loading control. D Immunoblotting of p-mTORSer2481, mTOR, p-p70S6KThr389, p70S6K, p-p62S349 and p62 in U87shC8#2 + C8WT and U87shC8#2 + C8Y380F cells. Vinculin was used as loading control. E Co-immunoprecipitation experiments (left) and relative quantification (right) of KEAP1 and p62 proteins in U87shC8#2 + C8WT and U87shC8#2 + C8Y380F cells. F Confocal microscopy analyses and G quantification of colocalizing dots in U87shC8#2 + C8WT and U87shC8#2 + C8Y380F cells; p62 (green), KEAP1 (red), DNA (Hoechst, blue); 4× digital magnification showing merged signals. H Representative immunofluorescence of U87shC8#2 + C8WT and U87shC8#2 + C8Y380F cells upon serum deprivation (-FBS); TFEB (green), DNA (Hoechst, blue); 4× digital magnification showing merged signals. I Quantification of TFEB staining of H reported as the ratio between nuclear and cytosolic fluorescence intensity (N/C). Results represent the mean of at least three independent experiments ( ± SEM). Statistical analyses: paired (C, E) and unpaired t test (B, G, I) (*P < 0.05; ***P < 0.001; ****P < 0.0001).
