Fig 2: Human iPS‐Neurons with Ndufs4KO are more susceptible to glutamate‐induced apoptosis, mediated through acetylation of p53. (A,B) Characterisation of human iPS‐Ns by immunofluorescence for pan‐neuronal marker β‐Tubulin (Tu‐20, red), vGAT (vesicular GABA transporter, green) or vGluT1 (vesicular glutamate transporter, green) and Hoechst (blue). (C) Morphology of mature human iPS‐neurons. (D,E) Representative fluorescence images of control and Ndufs4KO iPS‐neurons treated with glutamate (30 μM), NR (1 mM), and Ex‐527 (10 μM). (F) Quantitation of relative fluorescence intensity of indicated groups in (D,E) (log‐transformed for data normalisation). Data are mean ± s.e.m *p < .01, **p < .001, by ANOVA post hoc tests

Fig 3: PEPD binds directly to p53 mutants.a Recombinant human PEPD (10–2560 nM) was incubated with recombinant human p53 or p53 mutants (250 nM) overnight at 4 °C. Their binding was measured by ELISA. Each value is mean ± SD (n = 3). b Recombinant human PEPD (801 nM) and p53 (114 nM) or p53R175H (114 nM) were incubated alone or with each other for 15 min at room temperature, then incubated with BS3 (a cross-linker) for 30 min at room temperature, and analyzed by WB for PEPD, p53 and p53R175H. p53 was included in each experiment for comparison.

Fig 4: K373 acetylation is required for refolding and mitochondrial enrichment of p53 mutants freed from PEPD.a, b Cells were treated by siRNA (10 nM) for 48 h with or without C646 (8 µM). WCL was subjected to IP by PAb1620 or PAb240, followed by WB analysis of the precipitate for p53 mutants. c, d MDA-MD-231 (p53KO) cells were transfected with p53R175H/K373R, p53R248Q/K373R, p53R273H/K373R, or p53R280K/K373R and 24 h later treated with siRNA (10 nM) for 48 h. WCL was subjected to IP by PAb1620 or PAb240, followed by WB analysis of the precipitate by p53 mutants and PEPD. A p53-containing sample (WCL of untreated CAL-51 cells) was used as positive control during WB in c. e, f MDA-MD-231 (p53KO) cells were transfected with p53R175H/K373R or p53R280K/K373R and 24 h later treated with siRNA (10 nM) for 48 h. p53 mutants in subcellular fractions was analyzed by WB. Lamin B, a-tubulin and VDAC were measured as loading controls and for ruling out cross contamination.

Fig 5: p53 mutants refold once separated from PEPD.a Recombinant p53 and p53R175H were subjected to IP by PAb1620 or PAb240, followed by WB analysis of the precipitate for p53 and p53R175H. b–f WCL of untreated cells in b, c, and e or WCL of cells treated with siRNA (10 nM) for 48 h in d and f were subjected to IP by PAb1620, PAb240, or isotype-matched IgG, followed by WB analysis of the precipitate for p53 mutants and PEPD. A p53-containing sample (WCL of untreated CAL-51 cells) was used as a positive control during WB in b. The relative level of each p53 mutant in d–f was measured by ImageJ.