Fig 2: BioID macroautophagy bait-bait interactome. (A) Schematic of the 39 BioID baits within the macroautophagy pathway, separated into their respective step in macroautophagy. (B) A bait-bait network was created based on a spring-embedded layout where edge-width is proportional to peptide counts. Bait-bait proximity interaction network based on a spring embedded layout where edge width is proportional to peptide counts. A total of 129 protein-protein interactions (PPI) between baits: 109 are previously reported (green arrows), 20 are novel uncharacterized PPI (red arrows), and one is a newly confirmed protein-protein interaction (yellow arrow). ATG8, Atg8-family proteins.

Fig 3: Cholesterol depletion alters starvation-induced autophagy dynamics.a U2OS cells were starved in EBSS and treated or not with 2.5 mM methyl-β cyclodextrin (MBCD) in DMEM ± 100 nM Bafilomycin A1 (BafA1) for the indicated times before being lysed and subjected to western blot analysis. b Quantification of LC3B-II band intensity relative to Actin and normalized to DMEM control. Error bars = SEM. Significance was determined using two-way ANOVA followed by Sidak’s comparisons test from n = 3 independent experiments. P value = 0.0002. c U2OS cells were treated or not with 10 µM atorvastatin (ATV) for 48 h before starvation in EBSS ± BafA1 for the indicated times. d Quantification of data in (c). as LC3B-II band intensity relative to Actin and normalized to DMEM control. Error bars = SEM. Significance was determined using two-way ANOVA followed by Sidak’s comparisons test from n = 3 independent experiments. P value = 0.0454. e U2OS cells were treated for the indicated times with either 2.5 mM MBCD in DMEM or EBSS without MBCD before western blot analysis with the indicated antibodies. f U2OS cells were treated with 2.5 mM MBCD in DMEM or EBSS for the indicated time before fixation, immunostaining for ATG13, and wide-field microscopy. Scale bar = 20 µm. g Quantification of ATG13 puncta per cell normalized to the control. Error bars = SEM. Significance was determined using one-way ANOVA followed by Dunnett’s comparisons test from n = 3 independent experiments, >500 cells per condition. P value = 0.0151, 0.0042, <0.0001, 0.0007. h Cells expressing EGFP-BATS were incubated in EBSS or serum-free DMEM supplemented or not with 2.5 mM MBCD for 1 h before fixation and wide-field microscopy. Scale bar = 20 µm. i Quantification of EGFP-BATS puncta in (h). Error bar = SEM. The number of EGFP-BATS puncta per cell was normalized to the DMEM control. Significance was determined using one-way ANOVA followed by Dunnett’s post test from n = 3 independent experiments, >500 cells per experiment. P value = 0.0166 and 0.0024. Source data are provided as a Source Data file.

Fig 4: Autophagic cell death in lung tissues after 6 h of CI preservation. The LC3B-positive cells were observed in TUNEL-positive cells. The nuclei in TUNEL- and LC3B-positive cells were round, which was consistent with autophagic cell death (arrow). The shrunken or fragmented DNA was observed only in apoptotic nuclei (arrowheads). Scale bar=50 µm in the upper panel images and 10 µm in the lower panel images. CI, cold ischemia; LC3B, microtubule-associated protein 1 light chain 3B; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Fig 5: Y. pestis infection induces mitophagy in a type III secretion system (T3SS)-dependent manner. (A) Electron micrographs of double-membrane structure engulfing mitochondria (phagophore or mitophagosome) in THP-1 cells infected with strain 201 (MOI = 40 for 1 or 4 h). Arrowheads indicate phagophores or mitophagosomes, M indicates mitochondria. Scale bar = 200 nm. (B) THP-1 cells were infected with the indicated strains (MOI = 40 for 4 h) or stimulated with CCCP (30 µM) for 4 h, then stained for LC3B (green) and mitochondrial marker protein COX IV (red). Nuclei were stained with DAPI. Representative confocal images from two independent experiments were shown. Arrowheads indicated the colocalization of LC3B with mitochondria. Scale bar = 10 µm. (C) Pearson's coefficient values for colocalization of LC3B and COX IV in (B). The average Pearson's coefficients ± SD were calculated for 50 cells from two independent experiments by ImageJ 1.53e software (National Institutes of Health). One-way ANOVA followed by Tukey’s multiple-comparison test was used to measure significance. *, adjusted P < 0.05 and ***, adjusted P < 0.001. (D) THP-1 cells untreated or pretreated with Wortmannin (1 µM) for 1 h were infected with strain 201 (MOI 40 for 4 h) and stained with MitoSOX Red. Representative confocal images from three independent experiments were shown. Scale bar = 50 µm. The relative % of the MitoSOX Red positive cells were determined (n ˜ 200). Data are reported as the mean ± SD from three independent experiments. One-way ANOVA followed by Tukey’s multiple-comparison test was used to measure significance. *, adjusted P < 0.05 and ***, adjusted P < 0.001. (E) THP-1 cells/mouse BMDMs untreated or pretreated with Wortmannin (1 µM) for 1 h were infected with strain 201 (MOI 40 for 4 h). Cytosolic mDNA relative to nuclear DNA was analyzed by qPCR. Data are reported as the mean ± SD from three independent experiments. One-way ANOVA followed by Tukey’s multiple-comparison test was used to measure significance. *, adjusted P < 0.05; **, adjusted P < 0.01; and ***, adjusted P < 0.001.