Fig. 4: DELE1-ISR pathway negatively regulates mitophagy via attenuating protein synthesis.

a HEK293T PRKN OE^HL cells expressing a NTC sgRNA, or a sgRNA targeting OMA1 (OMA1 KD) or HRI (HRI KD) were left untreated or treated with 0.6 or 1.25 ng/mL oligomycin for 24 hr, followed by measurement of mitophagy via flow cytometry. (mean ± s.d., n = 3 independently treated culture wells) b Immunoblots of ATF4, HSPD1, PINK1, COX IV, LC3, and HRI. NTC and HRI KD cells with PRKN OE^HL were left untreated or treated with 10 μM CCCP or 1.25 ng/mL oligomycin for 24 h. β-Actin serves as loading control. c Wild type (WT) or two clonal eIF2α^S49/52/A cells with PRKN OE^HL were left untreated or treated with 1.25 ng/mL oligomycin with or without 100 nM bafilomycin A (Baf), followed by measurement of mitophagy via flow cytometry. (mean ± s.d., n = 4 independently treated culture wells) d Immunoblots of ATF4, HSPD1, PINK1, COX IV, LC3, phosphorylated and total eIF2α in WT and eIF2α^S49/52/A cells following 1.25 ng/mL oligomycin for 24 h. β-Actin serves as the loading control. e Flow cytometry measurement of mitophagy in WT or two ATF4 KO clonal cell lines with PRKN OE^HL following treatment with 1.25 ng/mL oligomycin for 24 h in the presence or absence of bafilomycin A (Baf). (mean ± s.d., n = 3 independently treated culture wells) f Immunoblots of ATF4, HSPD1, PINK1, COX IV and LC3 and β-Actin in WT and two ATF4 KO clonal cell lines following 10 μM CCCP or 1.25 ng/mL oligomycin treatment for 24 h. β-Actin serves as the loading control. g NTC and DELE1 KD cells with PRKN OE^HL were treated with 10 μM CCCP or 1.25 ng/mL oligomycin in the presence of cycloheximide at 12 different concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1, 2, 4, 8,10 and 20 μg/mL) for 24 h followed by flow cytometry to measure mitophagy. Cycloheximide 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) h Measurement of the import of MTS^COX8-GFP11 in HEK293T cells with an NTC or DELE1 sgRNA (DELE1 KD) following treatment with 1.25 ng/mL oligomycin or 10 μM CCCP in the presence or absence of 100 ng/mL cycloheximide (CHX). MTS^COX8-GFP11 was induced simultaneously during mitochondrial toxins by adding 500 ng/mL doxycycline. (mean ± s.d., n = 3 independently treated culture wells) Two-way ANOVA test followed by Turkey’s multiple comparisons test (two-sided). **** adjusted p value < 0.0001. i Cell free mitochondrial protein import assay using mitochondrial isolated from DELE1 KD cells treated with oligomycin alone or cotreated with oligomycin and cycloheximide for 24 h. j Immunoblots of ATF4, HSPD1, PINK1, COX IV and LC3 and β-Actin in WT and DELE1 KD cells with PRKN OE^HL following 10 μM CCCP or 1.25 ng/mL oligomycin treatment for 24 h with or without 100 ng/mL cycloheximide (CHX). p precursor; m mature. β-Actin serves as the loading control. k NTC and DELE1 KD cells with PRKN OE^HL are treated with 10 μM CCCP or 1.25 ng/mL oligomycin in the presence of Torin1 at 7 different concentrations (0, 50, 100, 200, 250, 500 and 1000 nM) for 24 h followed by flow cytometry to measure mitophagy. Torin1 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) l. Measurement of the import of MTS^COX8-GFP11 in HEK293T cells with an NTC or DELE1 sgRNA (DELE1 KD) following treatment with 1.25 ng/mL oligomycin or 10 μM CCCP in the presence or absence of 250 nM Torin1. MTS^COX8-GFP11 was induced simultaneously with mitochondrial toxins by adding 500 ng/mL doxycycline. (mean ± s.d., n = 3 independently treated culture wells) Two-way ANOVA test followed by Turkey’s multiple comparisons test (two-sided). **** adjusted p value < 0.0001. m. Cell free mitochondrial protein import assay using mitochondrial isolated from DELE1 KD cells treated with oligomycin alone or cotreated with oligomycin and Torin1 for 24 h. n Immunoblots of ATF4, HSPD1, PINK1, COX IV and LC3 and β-Actin in NTC and DELE1 KD cells with PRKN OE^HL following 10 μM CCCP or 1.25 ng/mL oligomycin treatment for 24 h with or without 250 nM Torin1. p precursor; m mature. β-Actin serves as the loading control. o Measurement of the expression levels of different mitochondrial substrates fused with GFP at their c-terminus. p mtKeima reporter cells with or without overexpression of different mitochondrial substrates (MTS^Diablo-GFP, SCO1-GFP, TIMM50-GFP, SLC25A19-GFP, TIMM44-GFP), expressing an NTC or DELE1 sgRNA were left untreated or treated with 0.6 ng/ml or 1.25 ng/ml oligomycin, followed by measurement of mitophagy using flow cytometry. (mean ± s.d., n = 3 independently treated culture wells) Two-way ANOVA test followed by Turkey’s multiple comparisons test (two-sided). **** adjusted p value < 0.0001. Source data are provided as a Source Data file.