Extended Data Fig. 3: Monitoring lysosomal delivery of mitochondrial fragments in HEK293 cells expressing the mitochondria-targeted ER-phagy receptors IDR modules.
(a) CLSM images of mock-transfected cells exposed to BafA1 to stabilize the cargo delivered within degradative endolysosomes and chemically fixed for immunostaining. Mitochondria are labeled with anti-TOMM20, endolysosomes with anti-LAMP1. Scale bar: 10 µm, inset magnification: 4x. (b) Same as (a) in cells transfected with Mito-GFP chimera. (c-h) Same as (b) in cells exposed to BafA1 to stabilize the cargo delivered within LAMP1-positive degradative endolysosomes and expressing mitochondria-targeted IDR chimeras. (i) LysoQuant quantification of the percentage of cellular endolysosomes that contain TOMM20-positive fragmented mitochondria in panels a-h (N = 3 biological replicates for a-d, N = 2 for e-h, n = 15, 28, 38, 41, 36, 20, 21, and 24 cells, for a-h, respectively). Ordinary one-way ANOVA with Turkey’s multiple comparisons test, F = 149.6. Mean bar is shown. Adjusted P value: ****p < 0.0001, ns (P = 0.9960), not significant. (j) Mitochondria-targeted ER-phagy receptors IDR modules interact with LC3B-II through their LC3-interacting motif. Co‐immunoprecipitation of GFP-tagged IDR modules (bait, upper panel) with LC3B (lower panel) from WT MEF cells treated with 50 nM BafA1 for 6 hours. LC3B-II interacts with IDR modules containing active LIR domains (lanes 2, 4, and 6). Lane 9: detergent-soluble total cell extracts (TCE) is shown. N = 3 biological replicates for lanes 1-3 and 9, N = 1 for lanes 4-8.
