Fig. 2: Loss of p66Shc protects against EGFR inhibition-mediated apoptosis.
From: p66Shc deletion confers apoptotic resistance to loss of EGFR-ERK signalling in neural stem cells
A Effect of AG1478 concentration on cell viability after 24 h treatment in WT and p66KO NSCs in NSC growth media, assessed by trypan blue exclusion assay; mean ± SEM, n = 3 independent experiments. B Representative phase contrast images of WT and p66KO NSCs for the following conditions: NSC growth media 24 h (Control) or treated with 2 μM AG1478 (EGFRi) for 24 h and 48 h. Scale = 20 μm. C, D Flow cytometry analysis of annexin V and PI staining in WT and p66KO NSCs after 48 h in NSC growth media (Control) or treated with 2 μM AG1478 for 24 h and 48 h. Representative flow cytometry plots (C), and quantification of live (Q1), early apoptotic (Q2), and late apoptotic/necrotic cells (Q3/4) (D); mean ± SEM, n = 3 independent experiments. E, F IF analysis of native CytC and CCasp3 staining in WT and p66KO NSCs treated with 2 μM AG1478, using ×20 (left) and ×60 (right) magnification. The hatched box indicates the magnified area. Scale = 50 μm (E). Quantification of cells negative for CytC, positive for CCasp3, and live cells (positive for CytC, negative for CCasp3) (F); mean ± SEM, n = 3 independent experiments, each with at least three fields of view. G, H Western blot analysis of EGFR-mediated signaling proteins including AKT, STAT3, ERK, and SHC in WT and p66KO NSCs treated with DMSO vehicle (CTRL) or 2 μM AG1478 for 4 h, followed by 20 nM EGF stimulation for 30 minutes. Densitometric analysis of the western blots (H); mean ± SEM, n = 3 independent experiments. Statistics were obtained using two-way ANOVA: ns, p\(\ge\)0.05; *p < 0.05; **p < 0.01; ***p < 0.001.
