EMBO/SourceData huggingface dataset:
Figure 1Immunoblot analysis of LC3B and GABARAP lipidation in wild-type (WT) and ATG16L1-KO HEK293A cells, with or without stimulation for 2h as indicated. BafA1 stands for Bafilomycin A1. Gamma-Tubulin (γ-Tubulin) is loading control. Densitometry analysis of lipidated LC3B (LC3B-II) to γ-tubulin (normalized to untreated WT cells) from immunoblots in (A). Data information: All data presented as mean + SEM, from n = 3 independent experiments. P-value determined using two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant.LC3B/GABARAP lipidation in ATG16L1- and ATG5-KO HEK293A cells treated for 2h as indicated. Vinculin is loading control. Cell lysates were immunoblotted for the specified proteins. Ratio of LC3B-II to Vinculin quantified from immunoblots in (C) and normalized to MC3 treated ATG16L1 KO cells. Data information: All data presented as mean + SEM, from n = 3 independent experiments. P-value determined using two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant.Immunoblot analysis of LC3B and GABARAP lipidation from WT, ATG16L1-KO (L1-KO), ATG16L2-KO (L2-KO), TECPR1-KO (TEC-KO), ATG16L1/L2-DKO (L1/L2-DKO), ATG16L1/TECPR1-DKO (L1/TEC DKO), and ATG16L1/L2/TECPR1-TKO (L1/L2/TEC-TKO) PC-3 cells, treated as indicated for 2h, were immunoblotted for the indicated proteins. Vinculin is loading control. MRT68921 and SAR405 are ULK and VPS34 inhibitors, respectively. Data information: All data presented as mean + SEM, from n = 3 independent experiments. P-value determined using two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant.LC3B/GABARAP lipidation in ATG16L1-KO and ATG16L1/TECPR1-DKO HEK293A cells with or without TECPR1 rescue, treated as indicated for 2h. Cell lysates were immunoblotted against the indicated proteins. Vinculin is loading control. Ratio of LC3B-II to Vinculin quantified from immunoblots in (G) and normalized to ATG16L1-KO cells treated with MC3. Data information: All data presented as mean + SEM, from n = 3 independent experiments. P-value determined using two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant.
Figure 2LC3B/GABARAP lipidation in WT, ATG16L1/TECPR1-DKO HeLa cells with and without EGFP-TECPR1 or EGFP-ATG16L1β rescue, treated for 2h as indicated. Cell lysates were immunoblotted for the indicated proteins. γ-Tubulin is loading control. Ratio of LC3B-II to γ-Tubulin quantified from immunoblots in (A) and normalized to untreated WT cells. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).Confocal images of ATG16L1/TECPR1-DKO HeLa cells expressing mScarlet-LC3B, with or without EGFP-TECPR1, treated for 2h with Cy5-labeled MC3 particles (left) or LLOMe (right) as indicated. Scale bars: 10 µm.Confocal time lapse images of ATG16L1/TECPR1-DKO HeLa cells, showing localization of mScarlet-LC3B and EGFP-TECPR1, after treatment with 400 μM LLOMe. Scale bar: 10 µm.Immunoblot analysis of LC3B/GABARAP lipidation in ATG16L1-KO HEK293A cells, treated with 400µM LLOMe for indicated periods of time. γ-Tubulin is loading control. Ratio of LC3B-II to γ-Tubulin quantified from immunoblots in (E) and normalized to 2hr (120min) LLOMe treatment. (Data presented as mean + SEM, n = 4 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test).
Figure 3Time series of confocal images showing redistribution of EqtSM-mCherry (sphingomyelin (SM) probe) and EGFP-TECPR1 in ATG16L1/TECPR1-DKO cells with doxycycline-inducible (dox, 2μg/ml) expression of EqtSM-mCherry cells following treatment with 400µM LLOMe. Scale bar: 10 µm.LC3B/GABARAP lipidation in ATG16L1- and TECPR1-KO HEK293A cells with or without dox-inducible (2μg/ml) expression of nSMase2-mCherry, treated for 2h as indicated. Cell lysates were immunoblotted against the indicated proteins. γ-Tubulin is loading control. Ratio of LC3B-II to γ-Tubulin quantified from immunoblots in (B) and normalized to untreated ATG16L1-KO cells. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).Confocal images of ATG16L1/TECPR1-DKO HeLa cells expressing EGFP-TECPR1, with or without dox-inducible (2μg/ml) nSMase2 overexpression, treated for 2h as indicated. Scale bars: 10 µm. Beeswarm-Superplot displaying mean number of EGFP-TECPR1 dots per cell per image of data represented in (E) quantified by automated analysis using CellProfiler. Mean ± SEM from each independent experiment (3/group) presented as large data points, with the black line corresponding to the mean of means. Individual data points (small) corresponding to single images are color-coded and superimposed according to the biological replicate they originate from. Total cells per condition/images per condition: 952/30 (LLOMe treated - nSMase2) and 1056/30 (LLOMe treated + nSMase2) were analyzed. (P-values determined using Student's unpaired t test).Liposome co-sedimentation assay of the indicated recombinant proteins and liposomes with or without SM. S: supernatant; P: pellet. Liposome binding in (C) was quantified as percentage of total protein in the pellet. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).
Figure 4Immunoblot analysis of LC3B/GABARAP lipidation in ATG16L1-KO or ATG16L1/TECPR1-DKO HEK293A cells with or without rescue with TECPR1 (WT), TECPR1 W77A, TECPR1 W154A, TECPR1 W829A or TECPR1 F908A, and treated with 400 µM LLOMe for 2h. γ-Tubulin is loading control. Ratio of LC3B-II to γ-Tubulin quantified from immunoblots in (C) and normalized to ATG16L1-KO cells. Data information: All data presented as mean + SEM (in (D) n = 3 independent experiments. P-values determined using one-way ANOVA followed by Tukey's multiple comparisons test in D and H; ns= not significant.LC3B/GABARAP lipidation in ATG16L1/TECPR1-DKO HEK293A cells rescued with TECPR1 (WT), TECPR1 W77A, TECPR1 W829A or TECPR1 W77A/W829A (WAWA), and treated with 400 µM LLOMe for 2h. γ-Tubulin is loading control.Maximum intensity projection of confocal images of HeLaK cells expressing EGFP-TECPR1 (WT) or W77A/W829A (WAWA) mutant, with or without 400µM LLOMe for 30min. Nuclei were counterstained with Hoechst 33342 (in blue). Scale bar: 10μm Beeswarm-Superplot displaying mean number of EGFP-TECPR1 dots per cell per image of data represented in (G) quantified by automated analysis using CellProfiler. Mean ± SEM from each independent experiment (3/group) presented as large data points, with the black line corresponding to the mean of means. Individual data points (small) corresponding to single images are color-coded and superimposed according to the biological replicate they came from. Total cells per condition/images per condition: 2502/59 (WT untreated), 2549/48 (WT LLOMe), 2256/59 (WAWA untreated) and 2816/59 (WAWA LLOMe) were analyzed.
Figure 5Coomassie-stained gel depicting in vitro LC3B lipidation reactions using the indicated combinations of proteins and liposomes (with or without SM), after being subjected to a liposome co-sedimentation assay. LC3B-AMP is denoted with an asterix (*). The extent of LC3B-I to -II conversion in (A) was determined and plotted as percentage of total LC3B. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).Liposome co-sedimentation assay of the indicated recombinant proteins and liposomes with or without SM. S, supernatant; P, pellet. Liposome binding in (C) was quantified as percentage of total protein in the pellet. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).Liposome co-sedimentation assay of the indicated recombinant proteins and liposomes with or without SM. S, supernatant; P, pellet. Liposome binding in (C) was quantified as percentage of total protein in the pellet. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).
Figure 6LC3B/GABARAP lipidation in WT, ATG16L1/TECPR1-DKO, ATG16L1-KO and TECPR1-KO HeLa cells with or without dox-inducible (2μg/ml) expression of mCherry-SopF, treated as indicated for 2h, were immunoblotted for the indicated proteins. γ-Tubulin is loading control. MRT68921 and SAR405 are ULK and VPS34 inhibitors, respectively. Ratio of LC3B-II to γ-Tubulin or Vinculin quantified from immunoblots in (A) and normalized to WT monensin treated cells. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant). (Data has skewed distribution. P-values were determined with Kruskal-Wallis followed by Dunn's multiple comparisons test).LC3B/GABARAP lipidation in WT, ATG16L1/TECPR1-DKO, ATG16L1-KO and TECPR1-KO HeLa cells with or without dox-inducible (2μg/ml) expression of mCherry-SopF, treated as indicated for 2h, Cell lysates were immunoblotted for the indicated proteins. γ-Tubulin is loading control. MRT68921 and SAR405 are ULK and VPS34 inhibitors, respectively. Ratio of LC3B-II to γ-Tubulin or Vinculin quantified from immunoblots in (A) and normalized to WT monensin treated cells. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant). (Data has skewed distribution. P-values were determined with Kruskal-Wallis followed by Dunn's multiple comparisons test).Confocal images of WT and TECPR1-KO cells with or without dox-inducible (2μg/ml) expression of mCherry-SopF, treated as indicated for 2h. Nuclei were counterstained with Hoechst 33342 (in blue). Scale bar: 10μm Beeswarm-Superplot displaying number of LC3 puncta per cell in the images represented in (E) quantified by automated analysis using CellProfiler. Mean ± SEM from each independent experiment (3/group) presented as large data points, with the black line corresponding to the mean of means. Individual data points (small) corresponding to single cells are color-coded and superimposed according to the biological replicate they originated from. Total cells per condition/images per condition: 1169/30 (WT LLOMe), 1064/29 (WT-mcherrySopF LLOMe), 692/23 (TECKO LLOMe) and 983/30 (TECKO-mcherrySopF untreated) were analyzed. (Data has skewed distribution. P-values were determined with Kruskal-Wallis followed by Dunn's multiple comparisons test).
Figure 1A Analysis of published single-cell RNA sequencing data from lungs of non-infected mice and mice infected with SARS-CoV-2 MA10 (GEO accession No. GSE186360). Av Mφ = alveolar macrophages, DC = dendritic cells, infl mo = inflammatory monocytes, non clas mo = non-classical monocytes, trans mo = transitional/intermediate monocytes.D Viral titers in lung homogenates at the indicated days post infection from mice infected as in B (bars/error bars represent mean ± SD, data points represent distinct biological samples, data points at the x axis represent samples below the limit of detection, day 3 data from two independent experiments, day 2 and 5 data are from single experiments, **p < 0.01, Mann-Whitney test).E IL-6 levels from lung homogenates and TNFα and IL-1β levels from serum on day 3 post infection (bars/error bars represent mean ± SD, data points represent distinct biological samples, data from two independent experiments, ***p < 0.001, t-test).F Viral titers in extrapulmonary organs on day 3 post infection were measured (bars/error bars represent mean ± SD, data points represent distinct biological samples [n=7 for heart, spleen, brain; n=5 for kidney and liver], data points at the x axis represent samples below the limit of detection, ns, multiple unpaired t-tests).G Representative lung sections from WT or IFITM3 KO animals stained for SARS-CoV-2 N protein. Scale bars represent 2 mm for whole lung images. Colored boxes overlaying the whole lung images correspond to the displayed magnified regions and scale bars in these images represent 0.2 mm. Red triangles, infected airways; solid black arrow, non-infected blood vessels; open black arrow, infected blood vessels.
Figure 2K18-hACE2 (hACE2) and K18-hACE2/IFITM3 KO mice were infected with 104 TCID SARS-CoV-2 WA1. A Weight loss (data points represent mean and error bars represent SEM, data from two independent experiments [hACE2 n=7, hACE2/IFITM3 KO n=8], skull/crossbones indicates that KO mice did not survive beyond this timepoint, *p < 0.05, ** p < 0.01, ANOVA with Bonferoni's multiple comparison's test).K18-hACE2 (hACE2) and K18-hACE2/IFITM3 KO mice were infected with 104 TCID SARS-CoV-2 WA1. C Viral titers in extrapulmonary organs on the indicated days post infection were measured (bars/error bars represent mean ± SD, data points represent distinct biological samples, day 3 data for heart, spleen, and brain are from two independent experiments, day 3 data for liver and kidney and day 5 data are from single experiments, data points at the x axis represent samples below the limit of detection, **p < 0.01, Mann-Whitney test).K18-hACE2 (hACE2) and K18-hACE2/IFITM3 KO mice were infected with 104 TCID SARS-CoV-2 WA1. D Representative lung sections from WT mice infected with SARS-CoV-2 MA10 (105 TCID) or K18-hACE2 or K18-hACE2/IFITM3 KO mice infected with SARS-CoV-2 WA1 (104 TCID) stained for SARS-CoV-2 N protein at day 5 post infection. Scale bars represent 2mm.
Figure 3WT and IFITM3 KO mice were intranasally infected with 105 TCID50 SARS-CoV-2 MA10 or mock infected with PBS (Non-Infected, NI). A Representative lung images from mice at the indicated times post infection were stained with hematoxylin and eosin. Scale bars represent 2mm for whole lung images and 500um for zoomed images. B Staining quantifications from multiple mice are shown (bars/error bars represent mean ± SD, data points represent distinct biological samples, *p < 0.05, ANOVA with Bonferoni's multiple comparison's test).
Figure 4WT and IFITM3 KO mice were intranasally infected with 105 TCID50 SARS-CoV-2 MA10 or mock infected with PBS (Non-Infected, NI). A Representative lung images from mice at the indicated times post infection were stained with anti-CD45. Scale bars represent 2mm for whole lung images and 500um for zoomed images. B Staining quantifications from multiple mice are shown (bars/error bars represent mean ± SD, data points represent distinct biological samples, *p < 0.05, ANOVA with Bonferoni's multiple comparison's test).
Figure 5WT and IFITM3 KO mice were intranasally infected with 105 TCID50 SARS-CoV-2 MA10. RNA sequencing analysis was performed on RNA extracted from infected WT and IFITM3 KO lungs on day 2 post infection. D Relative gene expression heat maps for genes from the top two most significant GO Biological Processes as shown in C.
Figure 1A HEK 293 cells were transfected with mCherry-tagged Parkin and FLAG-tagged UBQLN2 for 24 h, and then were treated with 5 µg/mL Antimycin A and Oligomycin A (A/O) for 4 h and 24 h. The cells were stained with anti-FLAG (green) antibody and anti-TOM20 (gray), and then were visualized using confocal microscopy. Scale bars, 10 µm. Insets were higher magnifications of the dashed box area. Scale bars, 2.5 µm. B, C The dashed square is magnified as shown in the boxed region in A with corresponding line scan indicating Parkin and UBQLN2 co-localization on the depolarized mitochondria (the line scan indicates pixel intensity of Parkin and UBQLN2).D HEK 293 cells were transfected with mCherry-tagged Parkin and EGFP-tagged UBQLN2. 24 h after transfection, cells were treated with 1 µg/mL A/O for 4 h, and then were harvested for the isolation of mitochondria. Whole cell (Total), cytosol (Cyto) and mitochondria (Mito) were lysed and analyzed by western blotting with the indicated antibodies.
Figure 2A Domain structure of Parkin in upper region. HEK 293 cells were transfected with HA-tagged UBQLN2 and FLAG-tagged Parkin WT or mutants (K161N, T240R, C418R, C431F and P437L) for 24 h, and then were treated with 5 µg/mL A/O for 5 h. The cells were stained with anti-HA (green) and FLAG (red) antibodies. Then the cells were visualized using confocal microscopy. Scale bars, 10 µm. Lower panel were higher magnifications of the dashed box area. Scale bars, 2.5 µm. B, C The quantification data of the Parkin or UBQLN2 with mitochondrial localization in A. At least 60 anti-HA-UBQLN2 or anti-FLAG-Parkin positive cells were counted for each sample. Data from three independent experiments were represented as means ± SD., ns, not significantly different; **, p<0.01; ***, p<0.001, one-way ANOVA.
Figure 3B HEK 293 cells were transfected with indicated siRNA. After 48 h, the cells were re-transfected with FLAG-Parkin and mt-mKeima for 17 h. Then the cells were treated with 5 µg/mL A/O for 5 h, and visualized using confocal microscopy. Intracellular mt-mKeima signal excited at 458 nm (measuring mitochondria with a neutral pH) was shown in green color, while red color indicated the mt-mKeima fluorescence excited at 561 nm (measuring mitochondria with an acidic pH) in the same cell. Scale bars, 10 µm.D The cells were transfected and treated as B. Cells were analyzed by FACS. The percentage of cells with lysosomal positive mt-mKeima are shown on the representative FACS data. Data from three independent experiments were represented as means ± SD., **, p<0.01; ***, p<0.001, one-way ANOVA.G HEK 293 cells were transfected with indicated siRNAs. After 24 h, the cells were re-transfected with FLAG tag or FLAG-tagged UBQLN2WT or ∆UBA, along with mCherry-Parkin for 24 h. The cells then were treated with or without 1 µg/mL A/O for 24 h. The cell lysates were subjected to immunoblot with indicated antibodies.
Figure 4A Domain structure of UBQLN2 in upper region. HEK 293 cells were transfected with FLAG-tagged UBQLN2-WT or ALS-linked mutants (∆UBA, ∆UBL, P497H or P509S), along with mCherry-Parkin. After 24 h, the cells were treated with 5 µg/mL A/O for 5 h. The cells were stained with anti-FLAG (green) and anti-TOM20 (gray) antibodies, and then were visualized using confocal microscopy. Scale bars, 10 µm. Insets were higher magnifications of the dashed box area. Scale bars, 2.5 µm.C The cells were transfected with mCherry-Parkin and HA-tagged UBQLN2-WT or ALS-mutants as A and subjected to immunofluorescent assay as in A, but were treated with 1 µg/mL A/O for 24 h. Scale bar, 10 µm. Yellow arrow indicates uncleared mitochondria.E HEK 293T cells were transfected with indicated siRNAs. After 24 h, the cells were re-transfected with FLAG-tagged UBQLN2-WT or mutants, along with mCherry-Parkin for 24h. Then the cells were treated with or without 1 µg/mL A/O for 18 h. The cell lysates were subjected to immunoblot with indicated antibodies.
Figure 5A HEK 293 cells were transfected with mCherry-tagged Parkin, HA-tagged UBQLN2, FLAG-tagged HSP70 and BFP-mito for 24 h, and were then treated with 5 µg/mL A/O for 4 h. The cells were stained with anti-HA (green) and FLAG (gray) antibodies, and were visualized using confocal microscopy. Scale bar, 10 µm. Right panel were higher magnifications of the dashed box area. Scale bars, 2.5 µm.D HEK 293T cells expressing HA-tagged UBQLN2 and FLAG-tagged HSP70 (WT or ΔC) were incubated with 5 µg/mL A/O for 5 h. The cells were then subjected to immunoprecipitation assay using anti-FLAG antibody.E HEK 293T cells expressing HA tagged UBQLN2-WT, ΔUBA or P497H) and FLAG-tagged HSP70 were incubated with 5 µg/mL A/O for 5 h. Immunoprecipitation assay were performed using UBQLN2 antibody.G The quantification data of UBQLN2 recruitment to mitochondria after 5 µg/mL A/O treatment. HEK 293T cells were transfected with indicated siRNAs. The cells were re-transfected with HA-tagged UBQLN2-WT, mCherry-Parkin, along with BFP-mito, and treated with 5 µg/mL A/O for 5 h. The cells were subjected to immunofluorescent assay using anti-HA antibody. Data from three independent experiments were represented as means ± SD., Sixty cells were counted for each sample. **, p<0.01, one-way ANOVA.
Figure 6A HEK 293T cells expressing mCherry-Parkin were pre-treated with proteasome inhibitor (20 µM MG132) for 12 h, and treated with 1 µg/mL A/O for 2 h. Then the cells were fractionated and proteins were immunoblotted for relative antibodies. B The level of Mfn2 (OMM protein), TOM20, COXIV and HSP60 was quantified and normalized relative to GAPDH, and was shown in A. The data from three independent experiments were presented as mean± SD., ns, not significantly different; **, p<0.01; ***, p<0.001, one-way ANOVA.HEK 293T cells were transfected with indicated siRNAs for 48 h. C The cells were re-transfected with mCherry-Parkin for 12 h and were pre-treated with or without 20 µM MG132 for 12 h and treated with1 µg/mL A/O for 2 h. The cell lysates were subjected to immunoblot with indicated antibodies. The data from three independent experiments were presented as mean± SD., ns, not significantly different; *, p<0.05; **, p<0.01, one-way ANOVA.HEK 293T cells were transfected with indicated siRNAs for 48 h. The cells were transfected with HA-tagged UBQLN2-WT or ∆379-462, along with mCherry-Parkin for 12 h. Then the cells were treated with 1 µg/mL A/O for 2 h. The cell lysates were subjected to immunoblot using indicated antibodies. G Relative densities from F were shown in G. The data from three independent experiments were presented as means ± SD., ns, not significantly different; *, p<0.05; **, p<0.01, one-way ANOVA.HEK 293T cells were transfected with indicated siRNAs for 48 h. H The cells were re-transfected with HA-tagged UBQLN2-WT, along with mCherry-Parkin for 12 h. Then the cells were pretreated with 20 µM MG132 for 12 h and treated with 5 µg/mL A/O for 4 h. Mitochondria isolated from cell lysates were treated with or without 10 µg/mL proteinase K (Pro.K) and then were subjected to immunoblot with indicated antibodies. Mito: the isolated mitochondria; Mito (Pro.K): mitochondria after Pro.K treatment. COXIV level in "Mito (Pro.K)" group was quantified and normalized relative to HSP60 in "Mito group". The data from three independent experiments were presented as mean± SD., ** p<0.01, ***, p<0.001, one-way ANOVA.HEK 293T cells were transfected with indicated siRNAs for 48 h. I The cells were re-transfected with HA, HA-UBQLN2-WT or P497H, along with GFP-Parkin and FLAG-PHB2 for 20 h, and then were treated with or without 5 µg/mL A/O for 4 h. Images of situ PLA using rabbit anti-LC3B and mouse anti-FLAG antibodies. Blue: nuclei (DAPI); white dots: PLA positive puncta. Scale bar, 10 µm. J Over 500 cells were counted for each sample in I. The data from three independent experiments were presented as mean± SD., *, p<0.05; ** p<0.01, ***, p<0.001, one-way ANOVA.