Fig 2: Inhibition of mitophagy does not oppose release of mtDNA-containing EVs. (a–d) MDA-MB-231 cells were transfected with siRNAs targeting ATG5 and ATG7 (siATG5/7; a and b), FIP200 (siFIP200; c and d), or a nontargeting control (siNT). Conditioned medium was collected from transfected cells over a 48-h period. EVs were purified from conditioned medium using differential centrifugation and analyzed by nanoparticle tracking. mtDNA was determined using qPCR with primers complementary to a sequence within the mitochondrial ND2 gene. Values represent the mean of three independent experiments; dotted line and error bars represent SEM. Cellular levels of ATG5/7 (a), FIP200 (c), and LC3B (a and c) were determined using Western blotting with actin or vinculin as a loading control. In a and c, cells were nutrient starved in Earle’s balanced salt solution (EBSS) where indicated before lysis.

Fig 3: The regulation of ER-phagy by ufmylation of CYB5R3.a Schematic representation of the CYB5R3-mCherry-GFP (CYB5R3-CG) reporter for monitoring lysosomal degradation of CYB5R3. b Fluorescence microscopic analysis. CYB5R3-CG or CYB5R3K214R-CG (KR) together with UFL1 and UFBP1 were co-transfected into CYB5R3-/- HeLa. Forty-eight hours after transfection, the cells were cultured under nutrient-rich or deprived conditions for 9 h in the presence or absence of Bafilomycin A1. The cells were fixed and observed by confocal microscopy. The number of single mCherry-positive punctae per cell was determined using a Benchtop High-Content Analysis System and CellPathfinder software without bias. The numbers of cells used to count the mCherry-positive punctae were 401, 226, 117, 136, and 129 from lanes 1 to 5. Bars: 10 µm. c Fluorescence microscopic analysis. CYB5R3-CG together with UFL1 and UFBP1 or UFBP1F196A V198A (FA, VA) were co-transfected into UFBP1-/- HeLa. The cells were analyzed as described in b. The numbers of cells used to count the mCherry-positive punctae were 335, 176, 90, 123, 114, and 102 from lanes 1 to 6. Bars: 10 µm. d Fluorescence microscopic analysis. CYB5R3-GFP together with UFL1 and UFBP1 were co-transfected into CYB5R3-/- HeLa. Forty-eight hours after transfection, the cells were cultured under nutrient-rich or deprived conditions for 9 h. The cells were fixed, immunostained with anti-FIP200, anti-WIPI2, and anti-LC3 antibodies, and observed by confocal microscopy. Bars: 10 µm. The number of punctae positive for GFP and FIP200, WIPI, or LC3 per cell (n = 30) was determined. e Fluorescence microscopic analysis. CYB5R3-CG together with UFL1 and UFBP1 were co-transfected into parental or ATG7-deficient HeLa cells. The cells were observed as described in b. The numbers of cells used to count the mCherry-positive punctae were 245, 150, 186, and 144 from lanes 1 to 4. Bars: 10 µm. For the box plots, horizontal bars indicate medians, boxes the interquartile range (25th–75th percentiles), and whiskers 1.5× the interquartile range; outliers are plotted individually. Data are means ± s.e. Statistical analysis was performed by Šidák’s multiple comparison test after one-way ANOVA. Source data are provided as a Source Data file.

Fig 4: Ferritin particles assemble to form liquid-like condensates. (A and B) Fluorescent images of the ferritin subunits FTH1 and FTL. WT and macroautophagy-deficient FIP200 KO HeLa cells stably expressing mGFP-FTH1 or mGFP-FTL were grown in DMEM (A) or treated with 10 µg/ml ferric ammonium citrate (FAC) for 24 h (B) and observed by fluorescence microscopy. Scale bars, 10 µm (main) and 1 µm (inset). (C) Immunoblots showing the expression levels of mGFP-FTH1 and mGFP-FTL in cells used in A. (D) TEM images of the ferritin condensates in FIP200 KO HeLa cells expressing GFP-FTH1. Scale bars, 500 nm (main) and 100 nm (magnified). (E) FRAP analyses of the ferritin condensates. WT HeLa cells expressing mGFP-FTH1 were treated with 100 µg/ml FAC for 24 h followed by 50 µM deferoxamine (DFO) for 5 h, and subjected to FRAP analyses. Scale bars, 10 µm (main) and 1 µm (magnified). (F) Quantification of E. Fluorescence intensities before photobleaching were set to 1. Representative results from two independent experiments are presented as means ± SEM (n = 5). (G) Coalescence of the ferritin condensates. WT HeLa cells expressing GFP-FTH1 were grown in DMEM and observed by time-lapse fluorescence microscopy at ~11-s intervals. Scale bars, 10 µm (main) and 1.5 µm (magnified). Source data are available for this figure: SourceData F1.

Fig 5: C9orf72 associates with the autophagy initiation complex. (a) Schematic of the LAP-tagged long C9orf72 isoform. (b) Immunoblotting of C9orf72 in an inducible stable cell line expressing a single copy of the long C9orf72 isoform. Lysates from MEFs generated from wild-type and c9orf72 knockout (c9 KO) mice were used to identify the correct immune-reactive band as endogenous C9orf72, as multiple bands were recognized by commercially available C9orf72 antibody. The LAP-tagged C9orf72 long isoform expresses at a comparable level to the endogenous protein. (c) Schematic of SILAC-affinity purification to identify C9orf72-associating proteins. Cell lines stably expressing the LAP-tagged C9orf72 were grown in isotopically ‘heavy’ medium containing 13C6,15N4-arginine and 13C6,15N2-lysine, while the parental line (i.e., no transgene) was grown in ‘light’ medium containing normal arginine and lysine. Elution after the PreScission cleavage step was used for mass spectrometry analysis. (d) Graphic representation of the C9orf72 interactome. Y-axis displays the average ratio of peptides identified in the heavy vs light labeling, while the X-axis represents individual proteins; a protein was considered to be C9orf72-associated if it was at least 8-fold above background signal. The protein ID, median SILAC ratio, and numbers of unique peptides identified are listed. (e) Confirmation of mass spectrometry results by IP-western blots, including RB1CC1, ULK1, ATG13, SMCR8 and WDR41. (f) Reciprocal immunoprecipitation using HA-tagged ATG13 and ATG101. Cells were transfected with plasmids as indicated with pcDNA6 as a control. HA-tagged ATG13 and ATG101 pulled down the LAP-tagged long C9orf72 isoform. (g) Schematic representation of in situ proximity-ligation assay. (h) Representative results for in situ proximity-ligation assay. Phalloidin staining, which labels F-actin, was used to outline the cells. Scale bar: 20 µm. (i) Quantification of in situ PLA results (N = 3, cell numbers >20 per experiment, ***, p < 0.001). C9, C9orf72.