Journal: The EMBO Journal

Article Title: A unified mechanism for mitochondrial damage sensing in PINK1-Parkin–mediated mitophagy

doi: 10.1038/s44318-025-00604-z

Figure Lengend Snippet: ( A ) Flow cytometry measurements performed as in (Fig. ). Left graph ns P = 0.0988, Right graph from left to right ns P = 0.5132, 0.2695, **** P ≤ 0.0001 (left graph exact P values from left to right P = 7e-15, 1.7e-14, 7e-15, 7e-15; right graph exact P values from left to right 4.62e-11, 7e-15, 7e-15) by two-way ANOVA with Dunnett’s multiple comparisons test. Error bars mean +/− SD. N = 6 independent experiments from two separate transductions (separate transductions denoted by open or closed circles). Error bars mean +/− SD. ( B ) Representative immunoblots of PINK1 stabilization and activity in HeLa dCas9-BFP-ZIM3 cells with indicated sgRNA +/− 10 µM CCCP for 4 h. N ≥ 3 independent experiments. ( C ) Volcano plots of PINK1-YFP (red) interactors measured by AP-MS as in (5I). CCCP treatment, where indicated, was overnight. Other samples were untreated. Two-sided Student’s t tests were performed. Values were corrected for multiple comparisons by calculating a FDR with the permutation method. Proteins were annotated as significant interactors if they had an absolute log2 fold change of >1 (black outline). Proteins associated with TOM (dark blue) and TIM23 (cyan) translocases and cytosolic chaperones (yellow) are indicated. N = 4 replicates/sgRNA on one occasion. The untreated control guide group was the same as used in (Fig. ). ( D ) CN-PAGE separated PINK1-YFP complexes visualized by in-gel fluorescence as in (Fig. ). N = 2 independent experiments with TOMM40 KD, one of which was with antibody gel shift. ( E ) Flow cytometry in HeLa PINK-YFP cells with doxycycline inducible expression of wild-type or C-terminal truncated TOMM5. Demonstrating rescue with TOMM5 WT but not TOMM5 ΔC. N = 6 replicates on at least two occasions. ns P = 0.4357, **** P ≤ 0.0001 (exact P values P < 1e-15 for all comparisons) by ordinary one-way ANOVA with Šídák’s multiple comparisons test. ( F ) Crystal structure yeast TOM complex (PDB: 6JNF (Araiso et al, )) demonstrating the location of TOMM5 and TOMM7 subunits. ( G ) LFQ proteomics of HeLa PINK-YFP whole lysates following the indicated knockdown/treatment. Two-sided Student’s t tests were performed. Values were corrected for multiple comparisons by calculating an FDR with the Benjamini–Hochberg procedure. Proteins were annotated as significant if they had an FDR < 0.05 and an absolute log 2 fold change of >0.5 (black outline). N = 4 replicates/sgRNA on one occasion. .

Article Snippet: TOMM5 WT or TOMM5 1–39 (truncation after residue 39) were cloned into the doxycycline inducible pSBtet-RN, a gift from Eric Kowarz (Addgene plasmid # 60503) (Kowarz et al, ).

Techniques: Flow Cytometry, Western Blot, Activity Assay, Protein-Protein interactions, Control, Clear Native PAGE, Fluorescence, Gel Shift, Expressing, Knockdown