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. 2021 Nov 11;48:102186. doi: 10.1016/j.redox.2021.102186

Fig. 6.

Fig. 6

PGAM5 mediates the KEAP1-dependent mitophagy. A. KEAP1 but not KEAP1 R380A/R415A suppresses PGAM5-induced Parkin translocation. PC6 cells were transfected with Parkin-EYFP, wt KEAP1 or KEAP1 R380A/R415A and PGAM5. **P < 0.01 and ****P < 0.0001, n = 6 dishes, 25 fields per dish, Ordinary one-way ANOVA followed by Holm-Sidak's multiple comparison test. B. KEAP1 but not KEAP1 R380A/R415A suppresses PGAM5-induced mitophagy. Primary cortical neurons were transfected with a mix of EGFP-LC3B and GFP-LC3C, mitochondrially targeted Kate2, wt KEAP1 or KEAP1 R380A/R415A and PGAM5. ***P < 0.001 and ****P < 0.0001, n = 8 dishes, 5 fields per dish, Welch's ANOVA followed by Dunnett's T3 multiple comparison test. C. PGAM5 E79A/S80A-induced Parkin translocation is insensitive to KEAP1. PC6 cells were transfected with Parkin-EYFP, wt PGAM5 or PGAM5 E79A/S80A and wt KEAP1. **P < 0.01 and ****P < 0.0001, n = 6 dishes, 20 fields per dish, Welch's ANOVA followed by Dunnett's T3 multiple comparison test. D. PGAM5 E79A/S80A-induced mitophagy is insensitive to KEAP1. Primary cortical neurons were transfected with a mix of EGFP-LC3B and GFP-LC3C, mitochondrial Kate2, wt PGAM5 or PGAM5 E79A/S80A and wt KEAP1. ***P < 0.001 and ****P < 0.0001, n = 8 dishes, 7 fields per dish, Welch's ANOVA followed by Dunnett's T3 multiple comparison test. E. KEAP1 silencing does not induce Parkin translocation in the absence of PGAM5. PC6 cells were transfected with Parkin-EYFP and scrambled shRNA, KEAP1 shRNA, or/and PGAM5 shRNA expressing plasmids. ****P < 0.0001, n = 6 dishes, 20 fields per dish, one-way ANOVA followed Holms-Sidak's multiple comparison test. Interaction between the treatment P < 0.0001, Two-way ANOVA. F. KEAP1 silencing does not induce mitophagy in the absence of PGAM5. Primary cortical neurons were transfected with a mix of EGFP-LC3B and GFP-LC3C, mitochondrial Kate2 and scrambled shRNA, KEAP1 shRNA or/and PGAM5 shRNA expressing plasmids. ****P < 0.0001, n = 8 dishes, 7 cells per dish, one-way ANOVA followed by Sidak's multiple comparison test. Interaction between the treatment P = 0.0003, Two-way ANOVA. G. KEAP1 silencing does not lead to mitochondrial loss in the absence of PGAM5. The neurons were transfected with neuronal marker hSyn-EGFP, mitochondrial DsRed2 and KEAP1 shRNA or/and PGAM5 shRNA and mitochondrial density was quantified at the end of the axon. **P < 0.01, n = 76–85 axons from 8 dishes, Kruskal-Wallis test followed by Dunn's multiple comparisons test.