EMBO/SourceData huggingface dataset:
Figure 7A Oxygen consumption rate (OCR) of cardiomyocytes from patient TAZ10 and control at basal conditions and after the administration of the indicated compounds.B Mitochondrial membranes analyzed by BN-PAGE and Western-blotting using indicated antibodies. For a loading control samples were separated on SDS gel and analyzed by Western-blotting (lower panel).C Western blot analysis of SDHA protein levels compared to VDAC3. Quantification of SDHA relative to VDAC3 in percent with control set to 100%. Error bars indicate range of values of two independent experiments.D Analysis of SDHA gene expression in triplicates by qPCR in cardiomyocytes, analyzed in (C).
Figure 1D. Representative immunofluorescence images of MEFs 48h after transgene induction stained for the indicated reprogramming factors. Scale bar represents 50 μm (n = 3 biological replicates).E. Log fluorophore expression superimposed on Uniform Manifold Approximation and Projection (UMAP) embedding of the dataset. Shown are fluorophore expression levels (gene exp.) on a logarithmic (ln) scale. F. Log transgene expression superimposed on a UMAP embedding of the dataset. Shown are transgene expression levels (gene exp.) on an a logarithmic (ln) scale.G. UMAP embedding of scRNA-seq dataset colored by technical replicate. Orange: Replicate 1, Blue: Replicate 2H. Cell cycle score superimposed on UMAP embedding of the dataset. See unsupervised analysis of single-cell RNA-seq section in Methods for further details.
Figure 5D. Log-normalized expression z-score of genes up- (top panels) and downregulated (bottom panels) by Ascl1 (left, blue violins) and MyoD1 (right, yellow violins) versus their scaled expression levels (x-axis) binned into 10% intervals. For each box, the centerline defines the median, the height of the box is given by the interquartile range (IQR), the whiskers are given by 1.5 * IQR and outliers are given as points beyond the minimum or maximum whisker.E. Fate titration plot of Ascl1 and MyoD1 double positive cells Shown are decision boundaries for indicated fates according to transcription factor levels. MyoD1 and Ascl1 expression shown on the x- and y-axis are log-normalized expression values scaled into the dynamic range of the single-positive condition Cells are colored according to their lineage endpoint as determined by CellRank.F. Representative immunofluorescence images of cells 3 days after transgene induction. Cells were stained for the fluorescent reporter present on the PiggyBac construct of the reprogramming factor. Arrows indicate multinucleated cells. Scale bar represents 50 µm. mutAscl1 = mutant Ascl1. G. Quantification of the percentage of cells with more than one nucleus among all transfected cells within a given condition. Data points represent biological replicates (n = 4). For each box, the centerline defines the median, the height of the box is given by the interquartile range (IQR), the whiskers are given by 1.5 * IQR and outliers are given as points beyond the minimum or maximum whisker. Pairwise comparisons performed with Mann-Whitney U test, correction for multiple testing performed with Benjamini-Hochberg correction. *: p < 0.05 (MyoD1 vs Ascl1: p = 0.03, MyoD1 vs mutAscl1: p = 0.03, MyoD1 vs Ascl1 & MyoD1: p = 0.03, MyoD1 vs mutAscl1 & MyoD1: p = 0.03, Ascl1 vs Ascl1 & MyoD1: p = 0.03, Ascl1 vs mutAscl1 & MyoD1: p = 0.03, mutAscl1 vs Ascl1 & MyoD1: p = 0.03, mutAscl1 vs mutAscl1 & MyoD1: p = 0.03
Figure 1(A) in vitro kinase assays were performed using purified recombinant kinases (ULK1 and ULK2) and substrate (ATG16L1) in the presence of radiolabelled ATP. ULK and ATG16L1 inputs were examined by western blot (WB) and substrate phosphorylation was analyzed by autoradiography (AR). Data information: Unless otherwise indicated experiments were performed three times.(B) Full-length or truncated versions of ATG16L1 were subjected to an in vitro ULK1 kinase assay. ULK1 and ATG16L1 inputs were examined by western blot and target phosphorylation by autoradiography. Data information: Unless otherwise indicated experiments were performed three times.(D) Full-length or mutated HA-ATG16L1 was purified from mammalian cells and subjected to an in vitro ULK1 kinase assay. Inputs were analysed by WB and target phosphorylation by AR. Data information: Unless otherwise indicated experiments were performed three times.(E) HEK293A cells were transfected with wild-type or phospho-dead ATG16L1 in the presence of wild-type or kinase-dead ULK1. Phosphorylation of ATG16L1 (S278 or S287) and inputs were examined by WB. Data information: Unless otherwise indicated experiments were performed three times.
Figure 2(A) Wild-type, ULK1/2 double knockout (dKO), or IKKα KO mouse embryonic fibroblasts (MEFs) were incubated with either complete medium, amino acid-deficient DMEM, or HBSS for 1 hour. Samples were immunoblotted using the indicated antibodies. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.(B) Wild-type, ULK1/2 dKO, or IKKα KO MEFs were infected with log phase Salmonella for 2 hours; bacteria-containing media was then removed and cells were incubated with gentamycin (50 µg/mL)-containing DMEM for 2 hours. Samples were immunoblotted using the indicated antibodies. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.(C) Wild-type, ULK1/2 dKO, or IKKα KO MEFs cells were infected with Salmonella for 1 hours. Autophagic capture of Salmonella was analyzed by immunostaining for LPS and LC3B. Representative images are shown (scale bars, 10 μm and 3 μm). Quantification was generated from 8 fields of view from a representative experiment. The experiments were repeated twice. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.(D) Wild-type, ULK1/2 dKO and IKKα KO MEFs were infected with Salmonella for 1 hour. Xenophagy rates were examined through Colony Forming Unit (CFU) assays. Quantification of infection rates by immunofluorescence is demonstrated in the right panel. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.
Figure 3(A) HEK293A cells were transfected with either flag-tagged WT ATG16L1 or T300A ATG16L1. ULK1 was co-transfected in increasing amounts where indicated. Cleavage of ATG16L1 was analyzed by WB of whole cell lysates. Levels of ATG16L1 cleavage were quantified from 3 biological repeats (right panel). Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.(B) HEK293A cells were transfected with either tagged wild-type, T300A, or S278/T300A ATG16L1 in the presence or absence of ULK1. Cleavage of ATG16L1 was analyzed by WB. Levels of ATG16L1 cleavage were measured from 3 biological repeats (right panel). Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.(C) HEK293A cells were transfected with the indicated plasmids in the presence or absence of a pan-caspase inhibitor Z-VAD-FMK (15 µM) for 4 hours. Cleavage of ATG16L1 was analyzed by WB of 3 biological repeats. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.(D) Wild-type or T300A-expressing HEK293A were treated with Salmonella in the presence or absence of ULK1/2 inhibitor (16 μM) for the indicated time points. Expression of ATG16L1 was analysed by WB. The experiments were performed twice.(E) ATG16L1 knock-out HEK293A cells transfected with the indicated HA GST ATG16L1 plasmids were infected with Salmonella for 1 hour. Xenophagy rates were examined through CFU assays. Quantification of infection rates by immunofluorescence is demonstrated in the right panel. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.
Figure 4(A) Wild-type MEF cells were infected with Salmonella for 25 minutes. Phospho-ATG16L1, total ATG16L1, and LPS were stained and analysed by immunofluorescence. Representative immunofluorescent images are shown (scale bars, 10 μm and 1 μm). Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.(B) Wild-type and ULK1/2 dKO were infected with Salmonella for 25 minutes. Immunofluorescence was performed using antibodies against LPS and ATG16L1. Representative immunofluorescent images are shown on the left panel (scale bars, 10 μm and 2 μm). Quantification of ATG16L1-positive bacteria from 7 fields of view from a representative experiment is shown in the right panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.(C) ATG16L1 knock-out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour. Bacteria were stained using anti-LPS antibodies to analyze localization in addition to ATG16L1. Representative immunofluorescent images of ATG16L1 and LPS are shown (scale bars, 5μm and 1 μm). Quantification of ATG16L1 localizing to bacteria from 7 fields of view from a representative experiment is shown in the lower panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.(D) ATG16L1 knock-out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour. Bacteria were stained using anti-LPS antibodies to analyze localization in addition to the autophagy marker LC3B. Representative immunofluorescent images of LC3B and LPS are shown (scale bars, 5μm and 1 μm). Quantification of bacteria undergoing autophagic clearance from 7 fields of view from a representative experiment is shown in the lower panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.
Figure 1(B) HEK293T cells transiently expressing FLAG and FLAG-tagged TEX264 were subjected to immunoprecipitation (IP). Inputs (20% of the lysates) and immunoprecipitates (from 80% of the lysates) were analyzed by immunoblotting using the indicated antibodies.(C) HEK293T cells transiently expressing WT or mutated TEX264-FLAG were subjected to immunoprecipitation with an anti-FLAG antibody and detected with the indicated antibodies.(D and E) MEFs stably expressing TEX264-GFP or its mutant were cultured in starvation media and immunostained with an anti-LC3 antibody. Bars: 10 µm and 1 µm (insets) (D). Quantification of the number of TEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 48 cells for each cell type (E).(F and G) WT and TEX264-KO HeLa cells (with or without the indicated TEX264 mutants with a C-terminal FLAG tag) stably expressing the ER-phagy reporter (ss-RFP-GFP-KDEL) were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, cells were cultured in starvation medium lacking amino acids and serum for 9 h (F). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± standard error of the mean (SEM) of three independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (G).
Figure 2(A and B) MEFs stably expressing TEX264-GFP were cultured in starvation media with or without bafilomycin A1 (Baf A1) and immunostained with anti-pTEX264 and anti-LC3 antibodies. Bars: 10 µm and 1 µm (insets) (A). Quantification of the number of pTEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 35 cells for each cell type (B).(C) HeLa cells were treated with 250 nM Torin 1 and Baf A1 for 16 h, and cell homogenates were subjected to an OptiPrep membrane flotation analysis.(D, E and F) WT, ATG3-KO, and FIP200-KO HeLa cells were cultured in starvation media lacking amino acids and serum with or without Baf A1 for 1 or 5 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies (D). Relative changes of the ratio of band intensities of phosphorylated TEX264 to HSP90 and total TEX264 during starvation are shown (E and F). Data represent the mean ± SEM of three independent experiments.
Figure 3(A) HeLa cells were cultured with 20 nM CK1 inhibitor (D4476) or CK2 inhibitor (CX4945) for 3 h, followed by starvation (with the inhibitors) for 3 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies.(B) Recombinant TEX264-FLAG was incubated with or without CK2 and the CK2 inhibitor (CX4945) in the presence of 200 µM ATP for 1 h.(C) HEK293T cells transiently expressing TEX264-FLAG and one of the three Halo-CK2A isoforms or Halo-CK2B were subjected to immunoprecipitation with an anti-FLAG antibody and detected with anti-Halo and anti-FLAG antibodies.(D and E) HEK293T cells transiently expressing WT or TEX264-FLAG mutants were subjected to immunoprecipitation (IP). Inputs (20% of the lysates) and immunoprecipitates (from 80% of the lysates) were analyzed by immunoblotting using the indicated antibodies (D). The band intensities in the IP fractions were quantified, and the phospho-serine:FLAG ratio was calculated. Data represent the mean ± SEM of five independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (E).(F and G) MEFs stably expressing TEX264-GFP were cultured with the CK2 inhibitor for 3 h followed by starvation (with the CK2 inhibitor) and immunostained with an anti-LC3 antibody. Scale bars represent 10 μm and 1 μm (insets) (F). Quantification of the number of TEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by an unpaired two-tailed Student's t-test. Data were collected from 85 cells for each cell type (G).(H and I) WT HeLa cells stably expressing the ER-phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, cells were cultured with 20 nM CK2 inhibitor for 3 h followed by starvation (with the CK2 inhibitor) for 6 h (H). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± SEM of six independent experiments. Differences were statistically analyzed by an unpaired two-tailed Student's t-test (I).
Figure 4(B) HEK293T cells transiently expressing WT or mutated TEX264-FLAG were subjected to immunoprecipitation with an anti-FLAG antibody and detected with the indicated antibodies.(C and D) MEFs stably expressing TEX264-GFP or its mutants were cultured in starvation media and immunostained with an anti-LC3 antibody. Bars: 10 µm and 5 µm (insets) (C). The intensity of the GFP signal under starvation conditions was quantified at TEX264-GFP puncta and the ER. The puncta:ER signal ratio was calculated. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 70 puncta for each cell type (D).(E and F) WT and TEX264-KO (expressing WT or TEX264-FLAG mutants) HeLa cells stably expressing the ER-phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, the cells were cultured in starvation medium lacking amino acids and serum for 9 h (E). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± SEM of four independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (F).
figf1(b) Density gradient fractions of NRK cells starved for 8 h were analysed by transmission electron microscopy (upper panel) and immunoblotted for LC3 or LAMP1 (lower panel). Proteins in fraction 1 were identified by mass spectrometry analysis. Three replicates were carried out from three independent purifications. Scale bar, 2 μm.(e) NRK cells grown on the SAMCell were transfected with FAM-RNAi by reverse transfection and the transfection efficiency was monitored by microscopy. Left panel, FAM-RNAi-positive cells; right panel, differential interference contrast (DIC) image of the cells in the left panel.
figf2(a) NRK cells stably expressing CFP-LC3; LAMP1-YFP were transfected with nonspecific (NS)- or Cltc (clathrin heavy chain)-RNAi and starved for 12 h. Scale bar, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.(c) Representative TEM micrographs of nonspecific- or Cltc-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bar, 5 μm.(d) NRK cell lines stably expressing LAMP1-Cherry; clathrin-GFP were starved for 0 or 4 h and observed by confocal microscopy. As a control, clathrin-GFP-expressing cells were starved for 0 or 4 h and then stained with MitoTracker red. Scale bar, 5 μm. The insets in a, c and d represent enlarged regions of interest. (e) Cells from d were analysed for Pearson's co-localization coefficent (R(r)). n = 30 cells from three independent experiments. Error bars indicate the s.d.(f) NRK cells stably expressing LAMP1-Cherry; clathrin-GFP were starved for 4 h and time-lapse images were acquired by spinning-disc microscopy. Scale bar, 500 nm. The white arrows indicate a clathrin punctum moving along a LAMP1-positive structure.(g) NRK cells were starved for 0, 4 or 6 h and then subjected to cell fractionation. Fractions were analysed by western blotting with antibodies against LAMP2 and clathrin heavy chain. 1 is the top fraction. The outline indicates the Lamp2-positive fraction.(h) Cell lines stably expressing clathrin-GFP were starved for 4 h, stained with antibodies against GFP and LAMP1, analysed by 3D-SIM. Scale bar, 2.5 μm. The right panels show enlarged regions of interest from the left panel: T, top view; L, lateral view; B, bottom view.(i) Left panel, representative TEM micrographs of purified autolysosomes from starved clathrin-GFP-expressing cells. Scale bar, 250 nm. Right panel, SIM image of purified autolysosomes stained with GFP and LAMP1 from the same experiment. Scale bar, 5 μm.(j) NRK or stable clathrin-GFP-expressing NRK cell lines were analysed by pre-embedding-immuno-electron microscopy using antibodies against GFP. Scale bar, 100 nm. (k) Quantification of the average number of gold particles per image from control or clathrin-GFP cells. n = 25 images from three independent experiments. The error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.
figf3(a) NRK cell lines stably expressing CFP-LC3; LAMP1-YFP were transfected with nonspecific (NS)- or PIP5K1B-RNAi and starved for 12 h. Scale bars, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.(c) Representative TEM micrographs of nonspecific- or PIP5K1B-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bar, 5 μm.(d) NRK cells were starved for 4 h, stained with antibodies against PIP5K1B and LAMP1, and then analysed by 3D-SIM. Scale bar, 2.5 μm. The right panels show enlarged regions of interest from the left panel: T, top view; L, lateral view; B, bottom view.(e) Fractions from Fig. 2f were analysed by western blotting with an antibody against PIP5K1B. The outline indicates the LAMP2-positive fraction.(f) NRK cells were starved for 4 h, autolysosomes were purified by density fractionation and the quality of purified autolysosomes was monitored by TEM (left panel). Right panel, purified autolysosomes stained with antibodies against PI(4)P and PtdIns(4,5)P2. Scale bar, 500 nm.(g) NRK cells were starved for 0 or 4 h and stained with antibody against PI(4)P. Scale bar, 5 μm.(h) NRK cells lines stably expressing OSBP-PH-GFP;LAMP1-Cherry were starved for 0, 4, or 8 h and observed by confocal microscopy. Scale bars, 5 μm.(i) NRK cell lines stably expressing OSBP-PH-GFP;LAMP1-Cherry were transfected with PIP5K1B-RNAi. At 60 h after transfection, cells were starved for 12 h and analysed by confocal microscopy. Scale bar, 5 μm.(j) NRK cell lines stably expressing LAMP1-Cherry;clathrin-GFP were transfected with nonspecific- or PIP5K1B-RNAi and starved for 12 h, and then observed by confocal microscopy. Scale bars, 5 μm. The insets in a, h-j represent enlarged regions of interest.(k) Cells from g were analysed for Pearson's co-localization coefficent (R(r)) by Olympus Fluoview. n = 30 cells from three independent experiments. Error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.