EMBO/SourceData huggingface dataset:
Figure 7Anti-hTAPBPL mAb (clone 54) neutralized the inhibitory activity of hTAPBPL-Ig on T cell proliferation Significance in was calculated by one‐way ANOVA with Dunnett test and others by two‐tailed Student's t‐test. * P<0.05, anti-hTAPBPL Ab group was compared with isotype Ab group.DBA/2J mice were injected s.c. with 1× 105 P388 murine leukemia cells, followed by injection of the anti-hTAPBPL mAb (25, 50, or 100µg) or isotype Ab (25, 50, or 100 µg) 3 times per week. Tumors size was measured over time. The mean tumor diameter (mm3) + S.D. at the indicated time points are shown.Tumor infiltrating CD4+ and CD8+ T cells were examined by immunofluorescence.
Figure 1LDH assay was performed after 24 h of cold stress on ice. Data are presented as mean ± SEM; n = 3, biological replicates. ****p < 0.0001, ***p < 0.001 by one-way ANOVA followed by Dunnett's multiple comparison test.
Figure 2Immunoblots of MICU1 and its mutants rescued by the lentivirus. Arrows indicate MICU1 or the dimer. * indicates the potential DTT-resistant dimer.
Figure 3MMP was monitored by TMRE fluorescence at every 1 h. FCCP (200 µM) and antimycin A (50 µM) were used as pretreatments for 30 min. Data were normalized by basal state of WT A549 cells (B-D) Data at 1 h (C), 5 h (D are presented as mean ± SEM; n = 4 (B-D) biological replicates, ****p < 0.0001, **p < 0.01, *p < 0.05 by paired T-test (C), or one-way ANOVA followed by Dunnett's multiple comparison test (DLDH release was measured after cold stress for 24 h. FCCP (200 µM), antimycin A (50 µM), mitoQ and decylQ (500 nM) were used as pretreatments for 30 min. Data are presented as mean ± SEM; n = 4 (G) , biological replicates, ****p < 0.0001, ***p < 0.001, **p < 0.01, by one-way ANOVA followed by Dunnett's or Bonferroni's multiple comparison test. whose raw value were normalized by the C11-BODIPY 581/591 without cold stress of non-treatment, are presented as mean ± SEM; n = 5, biological replicates. **p < 0.01, *p < 0.05, by one-way ANOVA followed by Dunnett's multiple comparison test.
Figure 4LDH release was measured after cold stress for 24 h. BAPTA-AM (10 µM) was used as pretreatments for 30 min. Data are presented as mean ± SEM; n = 3, biological replicates, ****p < 0.0001, by unpaired T-test.LDH release was measured after cold stress for 16 h. Data are presented as mean ± SEM; n = 5, biological replicates, *p < 0.05, by unpaired T-test.
Figure 1 B: Silver nitrate staining of proteins pulled-down using the control peptide (left) or the FFAT peptide (right), after SDS-PAGE. The two major differential bands are highlighted by arrows C: Tandem mass spectrometry result table showing the three top scored proteins identified in the FFAT peptide bound fraction. Score: protein score based on the sum of the ion scores of all peptides identified; Cov.; percentage of the protein sequence covered by identified peptides; Pep.: number of unique peptide sequences identified  E: Western blot analysis of proteins pulled-down using the control (Ctrl) peptide (left) or the FFAT (FFAT) peptide (right). The Input, FT (flow-through), and Elution fractions correspond to HeLa cell total protein extract, unbound proteins and bound proteins, respectively. Representative illustration of at least two independent experiments
Figure 5 A: List of the 5 top scored proteins interacting with MOSPD2 and known to contain a FFAT motif (see Appendix Table S1 for the full list). Proteins co-precipitated with GFP-MOSPD2 and GFP-MOSPD2 RD/LD proteins were identified by mass spectrometry; proteins were filtered for their ability to bind MOSPD2 and not the RD/LD mutant. The sub-cellular localization of the proteins is indicated [53,65-69]  B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagged STARD3 (B) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E) anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed using anti-Flag (B, D-F and anti-GFP (B-F) antibodies B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagge STARD3NL (C) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed usin anti-ORP1 (C), and anti-GFP (B-F) antibodies B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagge ORP1L (D Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E), anti-ORP1 (D) ), anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed using anti-Flag (B, D-F ), and anti-GFP (B-F) antibodies B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagge STARD11 (E Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E anti-GFP (B-F), and anti-actin (B-F) antibodies Immunoprecipitated material was analyzed using anti-Flag (B, D-F and anti-GFP (B-F) antibodies B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) an HA-tagged PTPIP51 (F) (WT and FFAT-deficient). Approximatively 5 µg of total protein extracts were analyzed by Western blot usin anti-HA (F) anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed using anti-Flag (B, D-F ), and anti-GFP (B-F) antibodies G: Immunoprecipitation (anti-Flag) experiment between Flag-tagged STARD3NL (WT and FFAT-deficient) and endogenous MOSPD2. Proteins extracts and immunoprecipitated material were analyzed by Western blot using anti-MOSPD2, anti-STARD3NL and anti-actin antibodies
Figure 9 A: Western blot analysis of MOSPD2, VAP-A and VAP-B proteins in control HeLa cells (WT) and in HeLa cells transfected with a control siRNA (siCtrl), and with siRNA targeting MOSPD2 (siMOSPD2), VAP-A and VAP-B (siVAP-A/VAP-B), and MOSPD2, VAP-A and VAP-B (siMOSPD2/VAP-A/VAP-B)  B-F: TEM images of control HeLa cell (B: HeLa -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm  B-F: TEM images of control HeLa cell C: HeLa/siCtrl -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm  B-F: TEM images o MOSPD2 (D: siMOSPD2 -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm  B-F: TEM images o VAP-A and VAP-B (E: siVAP-A/VAP-B -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm  B-F: TEM images o MOSPD2, VAP-A and VAP-B (F: siVAP-A/VAP-B/MOSPD2) -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm  G-I: Quantification by stereology of ER-mitochondria (G contacts. The percentages of mitochondria perimeter in contact with the ER (G are shown as means and error bars (SEM). (G) 200 (HeLa), 208 (siCtrl), 199 (siMOSPD2), 202 (siVAP-A/VAP-B), 199 (siMOSPD2/VAP-A/VAP-B) mitochondria from 8, 10, 8, 12 and 12 cells, respectively, were analyzed G-I: Quantification by stereology o ER-endosome (H contacts The percentages o ), endosome perimeter in contact with the ER (H are shown as means and error bars (SEM) (H, I) 202 (HeLa), 189 (siCtrl), 148 (siMOSPD2), 141 (siVAP-A/VAP-B), 221 (siMOSPD2/VAP-A/VAP-B) endosomes from 9, 19, 10, 14 and 12 cells, respectively, were analyzed. Kruskal-Wallis with Dunn"s multiple comparison test (*: P-values <0.05; ***: P< 0,001; ****: P <0.0001) G-I: Quantification by stereology o endosome-endosome (I) contacts. The percentages o endosome perimeter in contact with an endosome (I) are shown as means and error bars (SEM) (H, I) 202 (HeLa), 189 (siCtrl), 148 (siMOSPD2), 141 (siVAP-A/VAP-B), 221 (siMOSPD2/VAP-A/VAP-B) endosomes from 9, 19, 10, 14 and 12 cells, respectively, were analyzed. Kruskal-Wallis with Dunn"s multiple comparison test (*: P-values <0.05; ***: P< 0,001; ****: P <0.0001)
Figure 1All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.A) Immunohistochemistry analysis of small intestinal sections, using the indicated antibodies.All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.B) Western blot analysis with protein extracts obtained from ex vivo-purified intestinal crypts and probed with the indicated antibodies.All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.C) Haematoxylin and eosin staining of sections prepared from different intestinal tracts.All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.D) Immunohistochemistry analysis of small intestinal sections, using specific antibodies for the proliferation marker KI67.All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.E) Expression analysis by qRT-PCR of the proliferation related markers indicated in the figure using RNA extracted from intestinal crypts. Graphs show mean ± SD for three replicates.
Figure 2A) PAS and (B) Alcian blue staining of sections prepared from the different intestinal tracts isolated from AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.C) Quantification (mean ± SD) of Goblet cells in crypts and villi from the same mice sown in A and B. More than 100 crypts and villi were scored for each condition after 15 days from the first β-naphthoflavone administration.D) Immunohistochemistry analyses in AhCre Eed+/+ and AhCre Eedfl/fl mice injected with β-naphthoflavone and sacrificed after 15 days using a lysozyme-specific antibody (upper panels: LYZ, a Paneth cell-specific marker). Histochemical assay probing for alkaline phosphatase activity is presented in the middle panels (ALPI, an enterocyte-specific marker). Combined Ki67 immunohistochemistry and histochemical Alcian blue staining is presented in the lower panels (Alcian/KI67).E) GFP expression from an Lgr5-eGFPtransgene identifying LGR5+ ISCs in near-native agarose-embedded sections from Eed+/+ and Eed-/- mice at 30 days from the first β-naphthoflavone administration. Cell nuclei were counterstained with DAPI.
Figure 3A) Radiation-induced intestinal regeneration: AhCre Eed+/+ and AhCre Eedfl/fl mice were injected with β-naphthoflavone, irradiated with 10 Gy 15 days later, and sacrificed 4 and 8 days after irradiation. Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin (n=6).B) Immunohistochemistry analyses in AhCre Eed+/+ and AhCre Eedfl/fl mice treated as in A using H3K27me3 and KI67 specific antibodies at 8 days post-irradiation.C) Quantification (mean ± SD) of KI67 positive cells per crypt (B). More than 100 crypts were scored for each condition.D) In vitro organoids formation using crypts isolated from AhCre Eed+/+ and AhCre Eed-/- mice 15 days after the first β-naphthoflavone injection. Organoids pictures were taken 5 days later at low and higher magnification (insets).
Figure 4A) Volcano plot showing the expression changes with a minimal fold change of 3 (FC=3) at genome-wide levels in AhCre Eed-/- crypts relative to AhCre Eed+/+ crypts 15 days after β-naphthoflavone administration.B) Bar plot showing the percentage of H3K27me3-positive promoters among the differentially regulated genes in Eed-/- crypts. All gene promoters and 5 independent random sampling of 167 gene promoters extracted the mouse genome are presented as specificity controls.C) Intensity profiles for H3K27me3 and SUZ12 occupancy at the promoters of up-regulated genes with respect to down-regulated genes in Eed-/- crypts.D) Genomic snapshots of the Cdkn2a locus showing SUZ12 occupancy and H3K27me3 deposition at the promoter regions of both the p16 and the ARF isoforms.E) Genomic snapshots of the RNAseq profile of the Cdkn2a locus in AhCre Eed+/+ and AhCre Eed-/- crypts 15 days after β-naphthoflavone administration.F) Expression analysis by qRT-PCR (mean ± SD) of both Cdkn2a isoforms (p16Ink4A and p19Arf) in AhCre Eed+/+ and AhCre Eed-/- crypts 15 days after β-naphthoflavone administration.G) Gene set enrichment analysis (GSEA) using specific secretory and enterocytic transcriptional signatures with respect to the list of up-regulated genes in Eed-/- crypts.
Figure 5A) Haematoxylin and eosin (top panels) and PAS staining (bottom panels) of sections prepared from different intestinal tracts in AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice 15 days after the first β-naphthoflavone administration (n=5).B) Quantification (mean ± SD) of Goblet cells along the crypt-to-villus axis from the staining shown in A. More than 100 crypts and villi were scored for each condition.C) Immunhistochemistry analysis using anti-KI67 antibody of intestinal sections prepared from AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice 15 days after the first β-naphthoflavone administration.D) Radiation-induced intestinal regeneration: AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice (n=6) were injected with β-naphthoflavone, irradiated with 10 Gy 15 days later, and sacrificed 8 days after irradiation. Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin (first panels) and Alcian Blue (last panels). Sections were also stained for immunohistochemistry analysis (second and third panels) using H3K27me3 and Ki67-specific antibodies.
Figure 6A) Expression analysis by qRT-PCR (mean ± SD) of the indicated intestinal epithelial cell markers regulated by the Wnt and Notch signaling pathways that are involved in stem cell homeostasis, regionalization, and Goblet cell differentiation (n=3).B) Genomic snapshots of the indicated genomic loci for SUZ12 occupancy and H3K27me3 deposition in intestinal crypts of wild type mice (n=3).C) Expression analysis by qRT-PCR (mean ± SD) of the Goblet cell markers (Atoh1, Gfi1, Spdef), early secretory precursor cells (Dll1), NOTCH targets (Hes1, Olfm4) and intestinal stem cells (Lgr5, Rnf43) in wild type intestinal crypts purified 38hrs after treatment with the NOTCH inhibitor DBZ. DMSO served as vehicle negative control (n=4).D) ChIP analysis of Atoh1 and Gfi1 loci using H3K27me3 specific antibody. The same crypts described in C were used. H3K27me3 levels were normalized on Histone H3 density (percentage of H3 signal). Graphs show mean ± SD.
Figure 2(a) PPARγ mRNA expression in sperm from control and MSUS mice. Control n = 15, MSUS n = 13, two-tailed Student's t-test, P = 0.029, t = 2.30, df = 26.(c) Relative luciferase luminescence in transfected GC-1 cells exposed to serum from control or MSUS mice. Numbers correspond to serum from individual animals applied to different cell culture wells. Control n = 17, MSUS n = 22, two-tailed Student's t-test, P = 0.042, t = 2.1, df = 37.
Figure 3(b) Adult weight in the offspring of tesaglitazar-injected males (Tesa-inj, n = 16) compared to the offspring of vehicle-injected males (Vehicle-inj, n = 23). Two-tailed Student's t-test, P = 0.019, t = 2.44, df = 37.(c) Glucose level in the offspring of Tesa-inj (n = 14) and Vehicle-inj (n = 21) males during a glucose tolerance test. Repeated measures ANOVA, treatment effect P = 0.0083, F (1, 33) = 7.877, time effect P < 0.0001, F (4, 132) = 347.7, interaction P = 0.0776, F (4, 132) = 2.155 Conc.; concentration.
Figure 4(a Differentially expressed transposable elements in sperm from Tesa-inj and MSUS males. Data represent genes with P < 0.05 and similar fold change. Fold change in heat map represents log2(fold change) respective to the corresponding control group. Total overlap is presented in Extended Data Fig. 11.b) mRNAs/lincRNAs in sperm from Tesa-inj and MSUS males. Data represent genes with P < 0.05 and similar fold change. Fold change in heat map represents log2(fold change) respective to the corresponding control group. Total overlap is presented in Extended Data Fig. 11.(c,d) (c) Volcano plot and (d) heat map of differentially expressed (FDR < 0.05) mRNA/lincRNA in sperm from Tesa-inj males. Dashed black lines in (c) represent y = 1.3, equivalent to FDR = 0.05, and x = ±0.03, equivalent to FC = 1.23 and 0.81. Black dots represent top candidates, which all have FC > 2 or FC < 0.5, and FDR < 0.05. Teal dots represent non-significant genes.
Figure 5(b) Weight in adult male MSUS offspring and the offspring of males injected with MSUS serum compared to respective control groups. MSUS offspring n = 22, Control offspring n = 24, one-tailed Student's t-test (data reproduced) P = 0.045, t = 1.734, df = 44. MSUS serum-injected offspring n = 30, Control serum-injected offspring n = 31, two-tailed Mann-Whitney U = 334.5, P = 0.059.(c,d) Blood glucose levels in MSUS offspring and the offspring of MSUS serum-injected males following a 30-min restraint challenge. MSUS offspring n = 13, Control offspring n = 12, repeat measures ANOVA, treatment effect P = 0.016, F (1, 23) = 6.704, time effect P < 0.0001, F (3, 69) = 53.13, interaction P = 0.027, F (3, 69) = 3.25, at 15-min adjusted P = 0.0015, t = 3.693, df = 92. MSUS serum-injected offspring n = 17, and Control serum-injected offspring n = 14, repeat measures ANOVA, treatment effect P = 0.023, F (1,14) = 6.493, time effect P < 0.0001, F (3, 42) = 48.4, interaction P = 0.29, F (3, 42) = 1.29. Conc., concentration.
Figure 1B, C, D. WT, ∆cpt1∆ept1 (B), Δcho2 (C) or Δopi3 (D) cells expressing GFP-Atg8 were grown to logarithmic phase in SD-URA, and shifted to SD-N for 4 h of starvation. Cells were harvested at indicated time points and subjected to western blotting (left panel). Pgk1 was used as a loading control. Middle panel- Autophagic activity during starvation was quantified by calculating the ratio of free GFP to total GFP (GFP-Atg8 + free GFP). Statistical analysis was done by student's t-test (paired, two tailed) (ns- not significant, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments. Right panel- Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount, during SD and SD-N.E. WT, Δopi3, and Δcho2 as well as Δatg1 (negative autophagy control) cells expressing chromosomally-tagged Fba1-GFP were grown to log phase in SD, and starved in SD-N for 4 hours. Cells were harvested at indicated starvation time points and subjected to western blotting (left panel). Pgk1 was monitored as a loading control. Right panel- Autophagic activity during starvation was quantified, by calculating the ratio of free GFP to total GFP (Fba1-GFP + free GFP).F. WT, Δopi3, Δcho2 and Δatg1 cells on the background of pho8Δ60 pho13Δ strain, were grown to log phase in SD, and starved in SD-N for 4 hours. Pho8 activity was measured by the alkaline phosphatase assay.
Figure 2A. Representative images of GFP-Atg8 (green) and vacuole (red, stained with FM4-64). WT, Δopi3 and Δcho2 cells expressing GFP-Atg8 were grown to log phase in SD-URA medium and shifted to SD-N for 3 h. Cells were observed by confocal microscopy before (SD) and during nitrogen starvation (SD-N) (left panel). Scale bar 1 µm. Right panel- Quantification of of cells with or without GFP inside vacuoles. Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (****, p≤0.0001) (ns- not significant), error bars represent SEM of at least 3 independent experiments. Number of cells analyzed for each strain and condition: SD (WT (n=446), Δcho2 (n=272), Δopi3 (n=272)), SD-N (WT (n=398), Δcho2 (n=224), Δopi3 (n=213)).
Figure 3A, B. WT and Δopi3 cells expressing GFP-Atg8 were grown to log phase in SD-URA, and shifted to SD-N for 4 h, choline (1 mM) was not supplemented (-choline) or supplemented to SD and SD-N (+choline SD, SD-N) or only to SD-N (+choline, SD-N) as indicated. Cells were harvested at indicated time points and subjected to western blotting (left panel). Pgk1 was monitored as a loading control. Middle panel- Autophagic activity was quantified during starvation by calculating the ratio of free GFP to total GFP (GFP-Atg8 + free GFP). Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (ns-not significant, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments. Right panel- Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount in Δopi3 cells at growth (SD) or during starvation (SD-N), with choline supplemented as indicated. Statistical analysis was done by Sidak's multiple comparisons test compared to WT (ns-not significant, *, p≤0.05, **, p≤0.005, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments.C. Representative images of GFP-Atg8 (green) and vacuole (red, stained with FM4-64). WT and Δopi3 cells expressing GFP-Atg8 were grown in SD-URA and shifted to SD-N. Choline (1mM) was either excluded (-choline), added to growth and starvation medium (+choline SD, SD-N) or added only to starvation medium (+choline, SD-N). Cells were observed using confocal microscopy during starvation (left panel). Scale bar 1 µm. Right panel- Quantification of cells with GFP inside vacuoles. Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (****, p≤0.0001, ns- not significant), error bars represent SEM of 3 independent experiments. Number of cells analyzed for each strain condition; -Choline: WT (n=487), Δopi3 (n=316), Choline SD, SD-N: WT (n=251), Δopi3 (n=361), Choline SD-N: WT (n=320), Δopi3 (n=267). D. PC levels determined by shotgun lipidomics for WT and Δopi3 cells expressing GFP-Atg8, with or without choline (1 mM) supplementation to SD-N. Samples were taken after 4 h in SD-N medium.