Fig. 4: Notch-1 signaling regulates sensitivity to ferroptosis.

Densitometric quantification of Notch-1 in whole cell lysates from human iPSC-derived basal cholinergic neurons (a) and corresponding western blot (b). Densitometric quantification of LRP8 and GPX4 in HEK293 cells following treatment with Notch-1 specific siRNA or scrambled control siRNA (c) with the corresponding western blot shown in d. Densitometric quantification of LRP8, GPX4 and Notch-1 in Notch-1 CRISPR knockout (N1KO) and control (CTRL) MEF cell lysates (e) and a representative western blot shown in f. MTT cell viability assay of Notch-1 KO CRISPR knockout and CRISPR negative control MEFs following a 24 h incubation with a dose range of RSL3 (g) or erastin (h). Western blot of MEF cells incubated with RIN-1 or vehicle (CTRL) and probed with specific antibodies to LRP8, GPX4, Notch-1 and β-actin, as quantified in i with a representative western blot shown in j. MTT cell viability assays of MEF cells pre-incubated with RIN-1 (10 µM) or vehicle control (0.1% DMSO) for 48 h and following 24 h incubation with a dose range of RSL3 (k) or erastin (l). Quantification of LRP8 in WT and PS dKO MEFs 24 h post transfection with an NICD-containing plasmid or vector-only (mock) plasmid (m) and representative western blots demonstrating relative levels of LRP8, NICD, PS1, PS2 and β-actin (n). Densitometric analyses of proteins relative to β-actin are shown as mean values (±SEM) and individual points represent independent wells of cultured cells except in the iPSC neurons which are pooled from three wells. Abbreviations: HC = healthy control, sAD = sporadic Alzheimer’s disease, FAD PS1A246E = familial Alzheimer’s disease mutant presenilin 1 A246E, NICD = Notch intracellular domain, RIN-1 = RBPJ inhibitor-1; 2-(2-Fluorophenoxy)−4-(1-methyl-1H-pyrazol-5-yl) benzamide.