Fig. 3: Caspase-8 overexpression sustains p62-KEAP1 interaction, via mTORC1 activation. | Cell Death & Differentiation

Fig. 3: Caspase-8 overexpression sustains p62-KEAP1 interaction, via mTORC1 activation.

From: Caspase-8 expression and its Src dependent phosphorylation on Tyrosine 380 triggers NRF2 signaling activation in glioblastoma

Fig. 3

A Confocal microscopy analyses and B quantification of colocalizing dots in U87shCTR and shC8#1 cells; p62 (green), KEAP1 (red), DNA (Hoechst, blue); 4× digital magnification showing merged signals. C Co-immunoprecipitation and immunoblotting experiments (left) and relative quantification (right) of KEAP1 and p62 proteins in U87shCTR and shC8#1 cells; D Immunoblotting of p-p62S349 and p62 in U87shCTR and U87shC8 cells and relative densitometric analysis of p-p62S349 normalized on total p62. GAPDH was used as loading control. E Immunoblotting of p-mTORSer2481, mTOR, p-p70S6KThr389 and p70S6K in U87shCTR and shC8 cells and relative densitometric analysis of p-mTORSer2481 normalized on total mTOR. Vinculin was used as loading control. F Confocal microscopy analyses in U87shCTR and U87shC8 cells upon serum deprivation (-FBS) for 2 hours and treated with CQ 10 μM for 16 hours; p62 (green), KEAP1 (red), DNA (Hoechst, blue); 4x digital magnification showing merged signals. G Immunoblotting of p62, LC3 I and II in U87shCTR and U87shC8 cells upon serum deprivation (-FBS) for 2 hours and chloroquine (CQ) 10 μM for 16 hours. GAPDH was used as loading control. H Histogram representing the densitometric analysis of LC3 II normalized on LC3 I reported as the fold change between U87shCTR and shC8#1 cells upon chloroquine (CQ) treatment, as in (G). I Representative immunofluorescence (ApoTome) in U87-MG (left) and quantification (right) of GFP-LC3 signal in U87shCTR and U87shC8#1 cells stably expressing GFP-LC3-RFP-LC3ΔG probe, upon serum deprivation (-FBS) for 2 hours and chloroquine (CQ) 10 μM for 16 hours. The quantification of the same experiment carried out in U251shCTR and U251shC8#1 is also shown. GFP-LC3 (green), RFP-ΔLC3 (red), DNA (Hoechst, blue); 4x digital magnification showing merged signals. J Immunofluorescence of U87shCTR and U87shC8 cells upon serum deprivation (-FBS); TFEB (green), DNA (Hoechst, blue); 4× digital magnification showing merged signals. K Quantification of TFEB staining reported as the ratio between nuclear and cytosolic fluorescence intensity (N/C) in U87shCTR and U87shC8#1 cells. Results represent the mean of at least three independent experiments ( ± SEM). Statistical analyses: paired (C, D, E, H) and unpaired (B, I, K) t test (**P < 0.01; ***P < 0.001; ****P < 0.0001).

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