Fig. 5.

Endogenous PPM1H is up-regulated by different mitochondrial stressors and recruited to the organelle. (A) Representative immunoblot from PPM1H^WT MEFs treated with different mitochondrial and cell stressors for 24 h and (B) quantification of PPM1H/tubulin from N = 3 independent experiment. (C) Live cell imaging of PPM1H-mApple MEF expressing a GFP mitochondrial tag treated with or without oligomycin/antimycin either in normal medium (Left) or hypotonic buffer (Right) and (D) relative quantification of colocalization of PPM1H-mApple and GFP-mito. (E) Immunoblot analysis of whole cell extract, cytoplasmic fraction, and crude mitochondrial fraction in PPM1H^WT MEF after mitochondrial depolarization and (F) quantification of PPM1H levels in each of the three cellular compartments. O/A: Oligomycin/Antimycin (1/10 μM) Rot: Rotenone (0. 5 μM); Val: Valinomycin (2 μM); Iono: Ionomycin (5 μM); DA: Dopamine (10 μM); SS: Sodium selenite (7 μM); H₂O₂: Hydrogen peroxide (50 μM); AZD: AZD8055 (1 μM); MK: MK8722 (10 μM); Iver: Ivermectin (15 μM); DFP: deferiprone (1 mM); GTTP: Gamitrinib-triphenylphosphonium (5 μM); EBSS: Earle’s balanced salt solution. Each lane was loaded with 20 μg (15 μg for mitochondrial fraction) of protein lysates. Graphs show mean ± SEM from three independent experiments. For B–D, ordinary one-way ANOVA with Dunnett’s multiple comparison test versus DMSO (all compounds, black circles and *) or H₂O (SS, H₂O₂, DFP, and EBSS, black squares and #). For D, unpaired Mann–Whitney test and for F, ordinary two-way ANOVA with Sidak’s multiple comparison test from three independent experiments (normalized to whole cell DMSO control). (Scale bar, 50 μm.)