EMBO/SourceData huggingface dataset:
f1(a) Western blot analysis of CALM, actin, GAPDH and LC3-II in several cell lines (HeLa, HEK, CAD and murine embryonic fibroblast (MEF)) where CALM was knocked down using shRNA or siRNA, as indicated, or knockout with rescue experiment. In all experiments in this paper, we used a scramble siRNA or a luciferase shRNA as controls. The cells were starved in Hanks balanced salt solution (HBSS) and treated with Baf A1 as indicated. (BC, basal conditions; SE, short exposure; LE, longer exposure). Quantification of LC3-II/actin or GAPDH ratio is shown in Supplementary Fig. 1A. *Not specific.(b) LC3 dot counting in CALM knockdown cells. CALM was knocked down in HeLa cells expressing GFP-LC3 and with or without an siRNA-resistant form of wild-type CALM (rescue), as indicated. The cells were starved in HBSS for 4 h, fixed and subjected to fluorescence microscopy to score the number of LC3 dots per cell. The knockdown efficiency and the level of the siRNA-resistant form of CALM are shown on the left on the western blotting. The number of LC3 dots per cell (shown as mean ±s.d.) is shown on the graph for each condition (n≥300 cells per condition; BC, basal conditions). The ratio of the number of LC3 dots per cell between starvation and basal conditions is shown on the right (*P0.01; NS, not significant, two-tailed t-test). Pictures obtained from automated microscope are shown. Scale bars, 20 μm.
f2(a) p62 vesicle formation in CALM knockdown HeLa cells. Confocal pictures are shown. #CALM-downregulated cells where p62 vesicles accumulate. Data are representative of three independent experiments and shown as mean ±s.e.m. (n≥500 cells; *P0.01; two-tailed t-test). Scale bars, 5 μm.(b) Western blot analysis of p62, actin and GAPDH in HeLa cells and HEK cells (basal conditions, BC, or without serum for 1 h) where CALM was knocked down using shRNA or siRNA, as indicated. Data are mean ±s.d (n=3 experiments for HeLa cells and HEK cells; *P0.05; two-tailed t-test).(c) Percentage of huntingtinQ74-expressing cells with aggregates in CALM knockdown HeLa cells. Data depict one representative experiments performed in triplicate, out of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).(d) Tau-positive tangle formation in CALM knockdown cells. HeLa cells transiently expressing DsRed-tau 4R were treated with Baf A1 for 4 h as indicated. Cells were fixed and analysed by confocal microscopy after immnunostaining for phosphorylated tau using PHF1 antibody (green). Data represent the number of cells with phosphorylated tau-positive tangles as mean ±s.e.m. (n=3 experiments; *P0.01; two-tailed t-test). Scale bars, 5 μm.(b) Western blot analysis of p62, actin and GAPDH in HeLa cells and HEK cells (basal conditions, BC, or without serum for 1 h) where CALM was knocked down using shRNA or siRNA, as indicated. Data are mean ±s.d (n=3 experiments for HeLa cells and HEK cells; *P0.05; two-tailed t-test).
f3(a) Formation of endogenous ATG12 vesicles in CALM knockdown HeLa cells in basal (BC) and serum starvation conditions. Confocal pictures are shown with arrows indicating ATG12 vesicles in starvation conditions. Data are from one representative experiment, out of three independent experiments. Data shown as mean ±s.e.m. (n≥500 cells; *P0.01; two-tailed t-test). Scale bars, 5 μm.(b) Co-localization between GFP-ATG16L1 and internalized cholera toxin subunit B conjugated with Alexa555 (CTX; 20 min) in CALM knockdown HeLa cells. Confocal pictures are presented with magnified areas showing the co-localization between ATG16L1 and CTX in greater detail. Scale bars, 5 μm. The Pearson's coefficient between ATG16L1 and cholera toxin is shown. Data are representative of three independent experiments and shown as mean ±s.d. (n≥20 cells; *P0.05; two-tailed t-test).(c) Size of endogenous ATG12 vesicles in CALM knockdown HeLa cells in starvation conditions. Confocal pictures are shown with arrows indicating ATG12 vesicles and # indicating CALM knockdown cells. Data are representative of three independent experiments and shown as mean ±s.d. (n≥100 vesicles; *P0.05; two-tailed t-test). (AU, arbitrary unit). Scale bars, 5 μm.(d) Live-cell imaging of ATG16L1-GFP in CALM knockdown HeLa cells. ATG16L1 pictures from various time points of a 5-min movie are shown in inverted grey style. Arrows indicate ATG16L1 vesicles. The number of fusion events per 5 min is shown. Data are representative of five movies and shown as mean ±s.d. (*P0.05; two-tailed t-test). Scale bars, 5 μm.(e) In vitro fusion assay of post-nuclear supernatant from HeLa cells expressing either GFP-ATG16L1 or mStrawberry-ATG16L1 in control and CALM knockdown conditions. Confocal pictures are shown where ATG16L1-mStrawberry signal is shown in purple to enable better visualization. Fused vesicles appear in white. The ATP-negative condition, which prevents SNARE-dependent fusion, is also shown as a control for the reaction. Magnified areas are shown to allow visualization of the vesicles. The percentage of fused vesicles is represented. n=numbers of vesicles scored per field (a minimum of five fields were analysed per condition). Data are representative of two independent experiments and shown as mean ±s.d. (n≥100 vesicles). Scale bars, 5 μm. (*P0.05; two-tailed t-test).
f4(a) LC3 levels in CALM knockdown cells. CALM was knocked down in cells expressing or not expressing an siRNA-resistant form of CALM wild type (wt) or CALM 219 mutant, as indicated. The cells were lysed and subjected to western blotting. The knockdown efficiency, LC3-II levels and the level of the siRNA-resistant form of CALM are shown on the western blotting.(b) LC3 dots counting in CALM knockdown cells. CALM was knocked down in cells expressing GFP-LC3 with or without an siRNA-resistant form of CALM wild type (wt) or CALM mutant (219 mutant) as indicated. The cells were kept in full medium or starved in Hanks balanced salt solution (HBSS) for 4 h, fixed and subjected to microscopy to score the number of LC3 dots per cell. The ratio of the number of LC3 dots per cell (shown as mean ±s.d.) between starvation and basal conditions is shown on the graph (*P0.01; NS, not significant, two-tailed t-test; n≥300 cells per condition; BC, basal conditions).(c) Co-localization between ATG9A and VAMP3-HA or GFP-LC3 and VAMP8-HA in control and CALM knockdown HeLa cells stably expressing VAMP3-HA or VAMP8-HA. Confocal pictures are shown with magnified areas showing co-localization between ATG9A and VAMP3 or GFP-LC3 and VAMP8 in control cells and no co-localization in CALM knockdown cells. Quantification of ATG9A-VAMP3 or GFP-LC3-VAMP8 co-localization is shown on the graph as a Pearson's coefficient (data are mean ±s.d.; *P0.05; two-tailed t-test). Scale bars, 5 μm.
f5(a) Co-localization between ATG12 and VAMP2-HA in control and CALM knockdown HeLa cells stably expressing VAMP2-HA. Confocal pictures are shown with magnified areas on the right showing co-localization between ATG12 and VAMP2 in control cells and no co-localization in CALM knockdown cells. Quantification of ATG12-VAMP2 co-localization is shown on the graph as the Pearson's coefficient (data are mean ±s.d.;*P0.05; two-tailed t-test). Scale bars, 5 μm.(b) Western blot analysis of VAMP2, actin and LC3-II in HeLa cells where VAMP2 was knocked down, as indicated. The cells were starved in Hanks balanced salt solution (HBSS) and treated with Baf A1 as indicated. (SE, short exposure; LE, longer exposure.) Quantification of LC3-II/actin ratio is shown. Data are representative of three independent experiments and shown as mean ±s.d. (*P0.05; NS, not significant, two-tailed t-test).(c) LC3 dot counting in CALM and VAMP2 knockdown cells. CALM or VAMP2 were knocked down in cells expressing GFP-LC3. The cells were kept in full medium or starved in HBSS for 4 h, fixed and subjected to automated fluorescence microscopy to score the number of LC3 dots per cell. The number of LC3 dots per cell (shown as mean ±s.d.) is shown on the graph for each condition (n≥300 cells per condition; BC, basal conditions). (*P0.01; NS, not significant, two-tailed t-test).(d) Number of p62 dots per cell in VAMP2 knockdown. HeLa cells where VAMP2 was knocked down were fixed and subjected to microscopy after labelling endogenous p62 using specific antibody. The data represent the number of p62 dots per cell shown as mean ±s.d. (*P0.05; two-tailed t-test; n≥300 cells per condition).(e) Percentage of huntingtinQ74-expressing cells with aggregates in VAMP2 knockdown HeLa cells. Data are from one representative experiments performed in triplicate, out of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).(f) Western blot analysis of VAMP2, actin and LC3-II in HeLa cells stably expressing VAMP2-HA wild type or VAMP2-HA mutated in the CALM-binding site at different levels (wild-type clone 6, wt Cl6: low level; wild-type clone 11, wt Cl11: high level; mutant clone 11, AA Cl11: low level; mutant clone 12, AA Cl12: high level). The cells were treated with Baf A1 as indicated. (SE, short exposure; LE, longer exposure). Quantification of LC3-II/actin ratio is shown. Data are representative of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).
f6(a) Size of endogenous ATG12 vesicles in HeLa cells stably expressing either wild-type or mutant VAMP2. Confocal pictures are presented with magnified areas showing ATG12 vesicles. Data are representative of three independent experiments and shown as mean ±s.d. (n≥100 vesicles; *P0.05; two tail t-test). Scale bars, 5 μm.(b) Co-localization between ATG12 and LC3 in HeLa cells stably expressing either wild-type clone 11 or mutant clone 12 VAMP2. Confocal pictures are presented with magnified areas showing ATG12-LC3 co-localization. The Pearson's coefficient between ATG12 and LC3 is shown. Data are representative of three independent experiments and shown as mean ±s.d. (n≥100 vesicles; *P0.05; two-tailed t-test). Scale bars, 5 μm.(c) Size and number of endogenous ATG12 vesicles in VAMP2-knockdown HeLa cells in starvation condition. Confocal pictures are shown with arrows indicating ATG12 vesicles. Data are representative of three independent experiments and shown as mean ±s.d. for the size of ATG12 vesicle. (n≥100 vesicles; *P0.05; based two-tailed t-test). Scale bars, 5 μm.(d) Live-cell imaging of ATG16L1-GFP in VAMP2-knockdown HeLa cells. Confocal pictures from various time points of a 5-min movie are shown in inverted greyscale. Arrows indicate ATG16L1null. The number of fusion events per vesicles is shown. Data are representative of five movies and shown as mean ±s.d. (*P0.05; two-tailed t-test). Scale bars, 5 μm.(e) In vitro fusion assay of post-nuclear supernatants from control and VAMP2 knockdown HeLa cells expressing either GFP-ATG16L1 or mStrawberry-ATG16L1. Confocal pictures are shown where ATG16L1-mStrawberry signal is shown in purple to enable better visualization. Fused vesicles appear in white. The ATP-negative condition, which prevents SNARE-dependent fusion, is also shown as a control of reaction. Magnified areas are shown to allow visualization of the vesicles. The percentage of fused vesicles (white) is represented. Data are representative of two independent experiments and shown as mean ±s.d. (n≥100 vesicles). Scale bars, 5 μm. (*P0.05; two-tailed t-test). n=numbers of vesicles scored per field.
f7(a) Western blotting showing downregulation of lap (i) or increase in Atg8a-II (ii) expression level in adult fly heads on lap downregulation using the UAS-RNAi lines lapGD12732 or lapKK105767, or the heterozygous allele lap1. Quantification of lap/actin and Atg8a-II/actin is shown. Genotypes: Control w; elav-GAL4/+; for the RNAi lines: w; elav-GAL4/ lapKK105767 and w; elav-GAL4/+; lapGD12732/+; for the lap1 allele: w; elav-GAL4/+; lap1/+.(b) Western blotting showing the accumulation of tau in Drosophila adult flyheads on lap downregulation using the UAS-RNAi lines lapGD12732 or lapKK105767, or the heterozygous allele lap1. Quantification of tau/lapGD12732 is shown. Genotypes: Control w; elav-GAL4/+; for tau-WT: w; elav-GAL4/+; UAS-tau-WT/+; for RNAi lines: w; elav-GAL4/ lapKK105767; UAS-tau-WT/+ and w; elav-GAL4/+; lapGD12732/tau-WT; for the lap1 allele: w; elav-GAL4/+; lap1/+ or w; elav-GAL4/+; lap1/UAS-tau-WT.
f8(ai) Western blot analysis of tubulin, LC3-I and LC3-II in zebrafish larvae where ATG7 was downregulated, as indicated. (ii) Western blot analysis of CALM-HA, actin and LC3-II in zebrafishlarvae where CALM-HA was expressed, as indicated. The larvae were treated with ammonium chloride (NH4Cl), as indicated. (SE, short exposure; LE, longer exposure). Quantification of LC3-II/actin ratio is shown. Data are representative of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).(b) Modulation of autophagy alters Dendra-tau clearance dynamics. The fluorescence intensity of each individual cell was quantified at each timepoint (n≥ 31 cells, ≥9 larvae per treatment group) and mean cell intensity values for each drug treatment at each timepoint were calculated. Images were taken immediately after photoconversion and at 24, 30 and 48 h intervals thereafter. Rapamycin treatment significantly increased the rate of Dendra-tau clearance. Ammonium chloride (NH4Cl) treatment significantly decreases the rate of Dendra-tau clearance (**P0.01, ***P0.001, one-way analysis of variance (ANOVA)). Error bars are ±s.e.m.(c) Dendra-tau clearance in the presence of CALM: representative images of CALM with mosaic expression of Dendra-tau and full-length Dendra-tau taken immediately after photoconversion and at 6, 24, 30, 48 and 54 h after conversion. The fluorescence intensity of individual cells was quantified and mean fluorescent intensity of cells co-expressing Dendra-tau and either full-length CALM or Δ-ANTH CALM (larvae-ANTH-HA) constructs (n≥ 100 cells, ≥9 larvae per treatment group) at each time point was calculated. (i) Expression of full-length HA significantly delayed the clearance of CALM at all time points compared with Δ-ANTH CALM (***P0.001, one-way ANOVA). One graph, representative of three independent experiments, is presented. (CALM-ANTH-HA: 132 cells, 20 fishes; CALM-CALM: 18 cells, 8 fishes). Another two experiments are shown in Supplementary Fig. S5C. Error bars are mean ±s.d. (ii) Treatment with CALM alters the dynamics of CALM clearance. Expression of full-length CALM significantly delayed the clearance of CALM at all time points compared with Δ-ANTH CALM, as observed in i (P0.001, one-way ANOVA). However, treatment of Δ-ANTH CALM injected CALM with CALM slows the CALM clearance by 70% to a level comparable to that observed in larvae injected with full-length Dendra-tau. Ammonium chloride treatment of Dendra-tau injected with full-length null results in a modest (22%) decrease in Dendra-tau clearance, suggesting that CALM overexpression and ammonium chloride treatment have a cumulative effect (n≥25 cells, n≥9 Dendra-tau per treatment group). Error bars are mean ±s.d. Note that i and ii are distinct experiments.
f9(a) rho::GFP-tau fishes were incubated from 3 to 9 d.p.f. in either dimethyl sulphoxide (DMSO) or rapamycin (i) or from 3 to 7 d.p.f in EM alone or 10 mM ammonium chloride or 100 nM Wortmannin (ii). Images through the central retina at 9 d.p.f. (top panel; (i)) reveal rod degeneration (arrow) throughout the retina in control (DMSO-treated) larvae, whereas rod photoreceptors are present throughout the retina, particularly in the central region following treatment with rapamycin (arrow). Images taken through the central retina at 7 d.p.f. (bottom panel; (ii)) show normal photoreceptors in the marginal zones (arrows) and only limited numbers in the central region. NH4Cl exacerbates degeneration-photoreceptors are absent from the central retina and reduced/absent from marginal zones (arrows). Sections were stained with anti-rhodopsin (1D1) antibody. GFP labels whole rod photoreceptors, whereas rhodopsin is present in the rod outer segment. GFP co-localizes with the red rhodopsin label in all experimental conditions. Scale bars, 50 μm. Quantification of rodphotoreceptor degeneration (n=10 larvae per group; ***P0.001; ###P0.001, two-tailed unpaired t-test). Error bars are mean ±s.d.(b) Full-length CALM electroporation into rho::GFP larvae did not cause degeneration., while full-length CALM electroporation into rho::GFP-tau larvae exacerbated photoreceptor degeneration. Central retina sections in the region of the optic nerve head are presented. Scale bars, 50 μm. Data represent the ratio (in %) of the intensity of the GFP signal between CALM-HA electroporated eye versus control eye for five individuals per transgenic line. ***P0.05; two-tailed t-test. Error bars are mean ±s.d.(c) NH4Cl treatment of rho::GFP-tau immediately after unilateral CALM electroporation caused photoreceptor degenerationon the control (non-electroporated) side but did not alter the degeneration caused by CALM electroporation. Rapamycin treatment of Rapamycin::GFP-tau immediately after unilateral CALM electroporation rescued photoreceptors on the control (non-electroporated) side but not on the CALM-electroporated side. To demonstrate that loss of GFP corresponds to loss of photoreceptors, sections were stained with anti-rhodopsin (1D1) antibody. Wilcoxon signed rank test was used to compare the left eye versus the right eye of the same fish; Mann-Whitney test was used to compare drug treatment in the right eye of different fishes. *P0.05. Scale bars, 50 μm. Error bars are mean ±s.e.m.
f10(a) Histological section to demonstrate the individual cell layers of the zebrafish retina. The photoreceptor layer (PR, comprising rod and cone photoreceptors) lies immediately adjacent to the retinal pigment epithelium (RPE) at the outermost surface of the eye. Thioflavin-S labelling of retinal sections was used to identify neurofibrillary tangles in the photoreceptor layer (marked with yellow dotted lines). No labelling was observed in the retina of rho::GFP at 8 d.p.f., whereas distinct thioflavin-S-positive tangles (arrows) were observed in the photoreceptor layer of rho::GFP-tau fish. Note, the RPE is highly autofluorescent due to the presence of silver pigment. High power regions are shown in the top right of each panel.(b) Unilateral electroporation of CALM into the retina of rho::GFP-tau zebrafish resulted in a marked increase in thioflavin-S positive tangles in the electroporated retina in the photoreceptor layer (PR) compared with the control side. Top panel are lower magnification images to show the retinal cell layers. Thioflavin-S labelling is restricted to the PR layer. Note the RPE is highly autofluorescent due to the presence of silver pigment. Bottom panel are higher magnification images to show individual thioflavin-S tangles the largest of which are indicated by arrows.
Figure 1A LOUCY cells were treated in vitro with the indicated SMs with or without vincristine (VCR) for 48h. Apoptosis was determined by flow cytometry upon staining with Annexin-V and Propidium Iodide (PI). Mean ± SD of 3 independent experiments is shown; ** p <0.01, *** p <0.001 and **** p <0.0001 by unpaired Student's t-test.B Fresh primary leukemic cells from two pediatric patients, one with common B-ALL at relapse (left) and one with T-ALL at diagnosis (right), both with poor response to second line chemotherapy, were treated with LBW242 and VCR at the indicated concentrations. Apoptosis was measured after 24 h as described in A.The SM-drug combination activates caspases LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h. Cells were stained with respective fluorescent substrates for caspase-8 (C) and -9 (D) , and were subsequently analyzed by flow cytometry. Mean ± SD of 3 independent experiments is shown; * p <0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 by unpaired Student's t-test.The SM-drug combination activates caspases LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h. Cells were stained with an antibody detecting the active caspase-3 subunit (E), and were subsequently analyzed by flow cytometry. Mean ± SD of 3 independent experiments is shown; * p <0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 by unpaired Student's t-test.The SM-drug combination induces PARP cleavage. LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h. Cleavage of PARP was measured by Western blot using Actin as loading control (F). One representative immunoblot out 3 independent experiments is shown.
Figure 2A Necrostatin-1 (Nec) does not affect LBW-mediated sensitization to VCR-induced apoptosis. Apoptosis induction in LOUCY cells was determined by flow cytometry 48h after treatment with SM and/or VCR (left) and/or TNFα (right) in the presence or absence of the RIPK1-inhibitor Nec. Mean ± SD of 3 independent experiments is shown; * p <0.05 and ** p<0.01 by unpaired Student's t-test.B RIPK1 is dispensable for apoptosis induction by LBW242 and VCR. LOUCY cells were transfected with a non-targeting siRNA (neg-siRNA) or a RIPK1-specific siRNA. 6h post transfection, cells were treated with the indicated drugs and apoptosis was measured 40h thereafter. Mean ± SD of 3 independent experiments is shown; *** p<0.001 by unpaired Student's t-test. Knockdown (KD) was verified by Western blot at the time of apoptosis measurement and KD efficiency calculated compared to the control. One representative immunoblot out of 3 biological replicates is shown.C The SM-drug combination does not activate the canonical NF-κB pathway. Phosphorylation and degradation of IκBα upon treatment with VCR or TNFα and/or LBW242 was determined in LOUCY cells by Western blot following treatment with the indicated drugs for 5, 15 or 60 minutes using Actin as loading control. One representative immunoblot out of 3 independent biological replicates is shown.D KD of NEMO does not affect the SM-mediated sensitization to chemotherapy-induced apoptosis. Experiment was performed as described in B, except that an siRNA targeting NEMO was used. Mean ± SD of 3 independent experiments is shown; * p<0.05 by unpaired Student's t-test.E The SM-drug combination activates the non-canonical NF-κB pathway. Cleavage of p100 NF-κB2 to the p52 fragment was determined by Western blot 24h after treatment of LOUCY cells with chemotherapy or TNFα in combination with SM. Asterisk indicates an unspecific signal. One representative immunoblot out of 3 biological replicates is shown.F KD of RelB does not inhibit apoptosis. Experiment was performed as described in B, except that an siRNA targeting RelB was used. Mean ± SD of 3 independent experiments is shown; differences are not significant, by unpaired Student's t-test.
Figure 3A Expression of XIAP mRNA in samples obtained from BM of healthy individuals (Ctrl, n=10) or patients with AML (n=517) or ALL (n=306). Data are expressed as median, 25th and 75th percentile, whiskers indicate min/max; ** p<0.01; **** p<0.0001 by Games-Howell post-hoc test.B Protein levels of XIAP normalized to loading control in PDX ALL samples from pediatric and adult patients, analyzed by protein immunoassay (Simple Western) and depicted as mean (n=2, biological replicates); PBMC = peripheral blood mononuclear cellsParental and XIAP over-expressing NALM-6 cells (C) were treated with VCR apoptosis (Annexin-V / PI) was measured at the indicated time points (C) by flow cytometry. Mean ± SEM of 3 independent experiments is shown; **** p ≤0.0001 by unpaired Student's t-test. XIAP protein level was analyzed in parental and XIAP-overexpressing NALM-6 cells by protein immunoassay (Simple Western); Actin was used as loading control. One representative immunoassay out of 3 independent experiments is shown.D Parental and XIAP over-expressing REH cells (D) were treated with VCR or LBW242; apoptosis (Annexin-V / PI) was measured after 72h (D) by flow cytometry. Mean ± SEM of 3 independent experiments is shown; **** p ≤0.0001 by unpaired Student's t-test. XIAP protein level was analyzed by protein immunoassay (Simple Western); Actin was used as loading control. One representative immunoassay out of 3 independent experiments is shown.