Fig. 5: Modulation of mitophagy by small molecules that inhibit or activate the ISR.
a Measurement of mitophagy in WT HEK293T with PRKN OEHL treated with 1.25 ng/mL oligomycin and cotreated with ISRIB at 13 different concentrations for 24 h. (mean ± s.d., n = 3 independently treated culture wells) b Immunoblots of ATF4, HSPD1, PINK1, COX IV and LC3 and β-Actin in NTC and DELE1 KD cells with PRKN OEHL following 10 μM CCCP or 1.25 ng/mL oligomycin treatment for 24 h with or without 300 nM ISRIB. p: precursor; m: mature. White arrow heads indicate a faint band of precursor form of HSPD1 in NTC cells following co-treatment with oligomycin and ISRIB. β-Actin serves as the loading control. c NTC and DELE1 KD cells with PRKN OEHL were treated with 10 μM CCCP or 1.25 ng/mL oligomycin in the presence of halofuginone at 7 different concentrations (0, 2.5, 5, 10, 20, 40 and 80 nM) for 24 h followed by flow cytometry to measure mitophagy. Halofuginone concentrations were converted to their base-10 logarithmic values. A nonlinear regression analysis using a log(inhibitor) vs. response model with a variable slope (four parameters) was performed to generate the plot. (mean ± s.d., n = 3 independently treated culture wells) d Immunoblots of ATF4, HSPD1, PINK1, COX IV and LC3 and β-Actin in NTC and DELE1 KD cells with PRKN OEHL following 10 μM CCCP or 1.25 ng/mL oligomycin treatment for 24 h with or without 5 nM halofuginone (HF). p precursor; m mature. β-Actin serves as the loading control. e Model for how the ISR pathway regulates protein translation, mitochondrial protein import, PINK1 stability and mitophagy under varying mitochondrial stress conations. Created in BioRender. Guo, X. (https://BioRender.com/v19u792).
