EMBO/SourceData huggingface dataset:
Figure 3A. H295R/TR N-FlagFATE1 cells were transfected with D1 ER marker for ER (green) and mtRFP marker for mitochondria (red). Manders' coefficient for green-red signals colocalization in basal (white histograms) and Dox-treated (black histograms) cells is shown on the right (mean ± SEM; n=12). ***p<0.001. Scale bars, 10 μm.B. Effect of FATE1 expression on ER-mitochondria distance in H295R/TR N-FlagFATE1 cells measured using a split-GFP probe [29]. Quantification is shown in the graph. White histogram, number of fluorescent objects/cell in basal conditions; black histogram, number of fluorescent objects/cell in Dox-treated cells (mean ± SEM; n=6 with 90 cells analyzed in total). **p<0.01. Scale bars, 5 μm.C. Transmission electron microscopy images of H295R/TR N-FlagFATE1 cells cultured in basal conditions or treated with Dox. ER-mitochondria contacts are indicated by white arrowheads. Scale bars, 200 nm. Right, quantification of the number of contacts normalized by the number of mitochondria and the percentage of mitochondria with ER contact sites is shown in basal conditions (white histograms) and after Dox treatment (black histograms) of cells (mean ± SEM; n=38 for basal and 47 for Dox-treated cells). ***p<0.001.D. Transmission electron microscopy images of H295R/TRSF-1 cells cultured in basal conditions or treated with Dox. ER-mitochondria contacts are indicated by white arrowheads. Scale bars, 200 nm. Right, quantification of the number of contacts normalized by the number of mitochondria and the percentage of mitochondria with ER contact sites is shown in basal conditions (white histograms) and after Dox treatment (black histograms) of cells (mean ± SEM; n=41 for basal and 42 for Dox-treated cells). ***p<0.001.E. Mitochondrial shape (BacMam Mitochondria-RFP; in red) in H295R/TR N-FlagFATE1 cells shown by fluorescence confocal microscopy in basal conditions and after Dox treatment. FATE1 (green), DAPI (blue). A higher magnification of merged green and red signal is shown in the insets. Scale bars, 10 μm.F. Mitochondrial fragmentation index in H295R/TR N-FlagFATE1 cells after Dox treatment (red histogram) compared to cells cultured in basal conditions (white histogram) (mean ± SEM; n=111 for basal and 101 for Dox-treated cells). ***p<0.001.G. Mitochondrial fragmentation index in H295R/TRSF-1 cells after Dox treatment (green histogram) compared to cells cultured in basal conditions (white histogram). (mean ± SEM; n=37 for basal and 49 for Dox-treated cells). ***p<0.001.H. Mitochondrial membrane potential (∆Ψ) measured by TMRM fluorescence after Dox treatment of H295R/TR N-FlagFATE1 cells (red histogram) compared to cells cultured in basal conditions (white histogram). Mean ± SEM is shown. (mean ± SEM; n=3 with 12 replicates/experiment). ns, not significant.
Figure 4A. MitochondrialCa2+ uptake. H295R/TR parental clone (white/grey histograms) H295R/TRSF-1 (white/green histograms), H295R/TR N-FlagFATE1 (white/red histograms) and H295R/TRFATE1 cells (white/violet histograms) (mean ± SEM; n=3-5 with 12 replicates/condition). **p<0.01; ***p<0.001; ns, not significant.B. CytosolicCa2+ concentration. Histograms as in A (mean ± SEM; n=3-5 with 12 replicates/condition). ns, not significant.C. Mitochondrial (left) and cytosolic (right) Ca2+ concentrations in Dox-treated H295R/TRSF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs (mean ± SEM; n=3-5 with 12 replicates/condition). ***p<0.001; ns, not significant.D. Caspase 3/7 activity in H295R/TR N-FlagFATE1 cells cultured in basal conditions (white histograms) or in the presence of Dox (red histograms) treated with H2O2 (500 μM) C2-ceramide (50 μM) or staurosporine (STS) (1 μM). (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05; **p<0.01; ns, not significant.E. Caspase 3/7 activity in Dox-treated H295R/TRSF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs and treated with H2O2 or STS (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05; ns, not significant.F. Flow cytometry analysis of TUNEL staining in H295R/TR N-Flag FATE1 cells cultured in basal conditions or in the presence of Dox treated with vehicle (control) H2O2 (500 μM), C2-ceramide (50 μM) or staurosporine (STS) (1 μM).G. Caspase 3/7 activity in H295R/TR N-FlagFATE1 cells cultured in basal conditions (white histograms) or in the presence of Dox (red histograms) and treated with mitotane (50 μM) (mean ± SEM; n=5-8 with 3 replicates/condition). **p<0.01.H. Caspase 3/7 activity in Dox-treated H295R/TRSF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs and treated with mitotane (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05.
Figure 5A. Immunohistochemistry analysis of FATE1 expression in normal adrenal cortex and adrenal hyperplasia, adenoma and carcinoma (ACC). Testis tissue was used as positive control. Scale bars, 200 µm (low magnification); 50 μ m (high magnification).B. Table summarizing the relative intensity of FATE1IHC staining (H-score) in groups of normal, benign and malignant tissues. The highest FATE1 expression levels were found in ACC. n, number of samples studied for each category of tissues. NBST, normal and benign steroidogenic tissues.C. Prognostic parameters in ACC patients. Results are presented as hazard ratios (HR) including 95% confidence interval (CI). The significance level was set at α=5% for all comparisons. The results for SF-1, FATE1 and combined SF-1/FATE1 expression levels are shown both in univariate and mutivariate (adjusted for age, sex and ENSAT tumour stage) analysis.D. Overall survival of ACC patients according to FATE1 expression in their tumours, as measured by quantitative immunohistochemistry (low, green curve; high, red curve) in mutivariate analysis. p=0.008.
Figure 1A) Quantification of the effect of individual BCL-2-family proteins on HCT AKO cell death, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to at least three independent experiments, n>30 cells per condition per experiment. *** p<0,001 and * p<0,05 with respect to GFP condition.C) Western blot analysis of SMAC and cyt c release in GFP-BAX, tBID-GFP and GFP-BCLXL expressing cells at 12 h, 16 h and 24 h after transfection in HCT AKO. SN (supernatant, cytosolic fraction), P (pellet, membrane fraction) and PS (Ponceau Staining).F) Kinetics of the expression of tBID-GFP (blue) and PI intake upon tBID expression (purple) and unstransfected HCT116 AKO cells (in grey). Plots show the average fluorescence intensity (dark lines) and its standard deviation (shaded areas) from 3 technical replicates and 3 independent experiments. Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min.G) Quantification of PI+ cells from total transfected cells with GFP-BAX, tBID-GFP, GFP-BCLXL and GFP. n=3 independent experiments with >10000 cells per condition per experiment. *** p<0,001 with respect to untransfected condition.H) Cell death induced by tBID-GFP in HCT AKO cells in the absence and presence of ZVAD, NSA and Ferrostatin-1. Data correspond to three independent experiments, n>30 cells per condition per experiment. *** p<0,001 with respect to MOCK condition.
Figure 2A) Representative confocal immunofluorescence and bright field (BF) images of HCT DKO cells expressing tBID-GFP (in green) treated or not with COMBO (ABT737 + S63586). Cyt c shown in magenta and nuclei in cyan. Scale bar 10 µm. B) Effect of tBID-GFP and mCherry-BCLXL on cell death, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to at least three independent experiments, n> 30 cells per condition per experiment. *** p<0,001, ** p<0,025 and * p<0,05 with respect to tBID-GFP in HCT AKO condition.C) Representative confocal immunofluorescence images of HCT DKO cells upon indicated treatments. Nuclei (cyan) and cyt c (magenta) are shown. Scale bar 10µm. Arrowheads represent cells showing released cytochrome c and pyknotic nuclei.E) % of cell death induced by TRAIL (1µg/ml), COMBO and their combination at different treatment durations on HCT DKO measured as in panel B. Paired Student's t-test ** p<0,025 and * p<0,05 with respect to MOCK condition.I) Effect of TRAIL (1µg/ml), COMBO and their combination on HCT DKO and TKO cell death at different time points normalized to control cells. Each dot corresponds to the average of 3 technical replicates from 4 independent experiments. *** p<0,001.J) Representative HyVolution - superresolution images of TFAM-RFP expressing U2OS BAX/BAK DKO cells in the in the absence (top) and presence of tBID-GFP (bottom). TFAM appears in red, TOM20/mitochondria in grey and tBID-GFP in green.K) Quantification of cells with TFAM release on U2OS BAX/BAK DKO cells transfected with GFP-BAX/tBID-GFP and TFAM-RFP in the presence/ absence of COMBO. Dots correspond to individual experiments (n=3 individual experiments with more than 10 cells per condition per experiment). Unpaired Student's t-test *** p<0,001 with respect to non-treated conditions.
Figure 3B) Cell death induced by tBID-GFP and its variants on HCT AKO, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to three independent experiments, with n> 30 cells per condition per experiment. *** p<0,001 and * p<0,05 with respect to tBID-GFP WT condition.D) Effect of tBID wt and α6-mutants overexpression on HCT AKO cells, measured as DRAQ7+/GFP+ cells and normalized to tBID wt condition. n=3 independent experiments, ** p<0,025 and * p<0,05 with respect to tBID-GFP wt condition. E) Effect of tBID wt and α6-mutants overexpression on MEF BID KO cells, measured as DRAQ7+/GFP+ cells and normalized to tBID wt condition. Effect of GFP overexpression on BID KO cells relative to tBID wt was assessed by pyknotic nuclei formation quantification. n=3 independent experiments *** p<0,001 with respect to tBID-GFP wt condition.
Figure 4A) Representative CLEM images of mitochondria in HCT AKO cells (control) and HCT AKO cells expressing GFP-BAX or tBID-GFP. Black arrows indicate MOM disruptions. B-D) Effect of GFP -BAX and tBID-GFP on HCT AKO mitochondrial structure. (B) Number of disruptions at the MOM per mitochondrial area, each dot represents an individual cell. (C) Mean area per mitochondrion at the condition tested, each dot represents an individual mitochondrion. (D) Analysis of cristae morphology, each dot represents an individual crista. Cristae shape calculated as length/width. Data represent n>50 mitochondria from 3-5 independent cells. In B-D), boxes represent 96% confidence interval, the average is represented by the line inside the box and whiskers correspond to S.D. The statistical significance was assessed by unpaired Student's t-test. *** p<0,001 and ** p<0,025 with respect to HCT AKO or untransfected condition.E) Representative confocal and STED images of GFP-BAX and tBID-GFP expressed in HCT AKO. Scale bar 1 µm for BAX images, 5 µm for tBID images. F) Zoomed-in structures of GFP-BAX supramolecular assemblies with the shape of lines (*double lines), arcs and rings in HCT AKO cells. Scale bar 100 nm.
Figure 5A) representative western blot of cytosolic cyt c and SMAC in HeLa cells. B,C) Western Blot analysis of active BAX (B) and BAK (C), in HeLa cells treated with STS or infected with Shigella flexneri (MOI 30) at different time points after infection.E) Representative Western Blot analysis of cytosolic and total (lysate) cyt c and SMAC in HCT wt, HCT DKO and HCT TKO cells infected with Shigella flexneri at the indicated time points after treatment/infection.
Figure 6A) Caspase-3/7 activity induced by ABT199 and etoposide on Nalm6 (venetoclax-sensitive) and Nalm6 199R (venetoclax-resistant) cells, normalized to untreated cells. Unpaired Student's t-test *** p<0,001 and ** p<0,025 (Nalm6 with respect to Nalm6 199R at different conditions). Dots correspond to technical replicates of n=3 independent experiments.D) Representative kinetics of cell death, represented as DRAQ7+ cells in Nalm6 199R cells (grey), Nalm6 199R BID KO #1 cells (blue) and Nalm6 199R BID KO #5 cells (purple) upon 250 ng/ml TRAIL treatment. Plots show the average fluorescence intensity (dark lines) and its standard deviation (shaded areas) from 5 technical replicates. Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min.EH) Cell death induced by GFP-BAK and GFP-BAK R127H on HCT AKO, measured as DRAQ7+/GFP+ cells. Unpaired Student's t-test. *** p<0,001 with respect to GFP BAK wt.I) Effect of 24 h incubation with 250 ng/ml TRAIL on Nalm6 199R, Nalm6 199R BAK KO and Nalm6 199R BOK KO, measured as DRAQ7+ cells with respect of total cell surface and normalized to untreated conditions. Each dot corresponds to the average normalized value of 3-4 technical replicates in n=3 independent experiments.
Figure 1(I) Representative image showing changes in colon morphology and length following DSS-induced colitis with concurrent treatment with MBELNs. (J) Column graph showing changes in colon length. Data are mean ± SEM from seven biological replicates, **P<0.01 using one-way ANOVA. (Hematoxylin and eosin (HE) staining to show histological changes in colon tissue Scale bar 200 μm.(M) Enzyme-linked immunosorbent assay (ELISA) for interleukin (IL)-6 and IL-1β from colon tissue. Data are mean ± SEM from seven biological replicates, **P<0.01 using one-way ANOVA.
Figure 2(A) Mass spectrometry (MS) analysis of aryl hydrocarbon receptor (AhR) pulldown protein from Mulberry bark derived exosome-like nanoparticles (MBELNs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated MC38 cell lysate. Data from three technical replicates are shown.(D) Representative images showing effect of MBELNs with and without TCDD (1 nm)/ carbobenzoxy-Leu-Leu-leucinal (MG132; 20 µM) on co-expression of AhR and COPS8 using immunofluorescence. Scale bar 10 μm, images are from three biological replicates.(E) Western blot showing MBELNs, TCDD and MG132 effect on co-expression of AhR and COPS8. The data represent as mean ± SEM from three biological replicates **P<0.01, ***P<0.001 using one-way ANOVA.(F) Heat map showing MS analysis of AhR pulldown protein from MC38 cells treated with MBELNs in combination with proteasome inhibitor, MG132. Data are represented from three technical replicates.
Figure 3(C) Transmission electron microscopy (TEM) of crypts of COPS8fl/fl and COPS8ΔIEC mice. The base of the crypt in COPS8ΔIEC mice is occupied by poorly differentiated columnar epithelial cells that lack secretory granules, rudimentary electron-dense granules (black arrows), granules in the lumen (blue arrows) and have a contracted endoplasmic reticulum (ER) (white arrows). Scale bar 5μm, data are from seven biological replicates per genotype.(G-H) Real-time quantitative reverse transcription Polymerase chain reaction (PCR) (qRT-PCR) analysis of the expression of genes encoding AMPs in the ileum (G) and colon (H) in COPS8fl/fl and COPS8ΔIEC mice. Error bars indicate the mean ± SD from three biological replicates. *P<0.05, **P<0.01, ***P<0.001 using Student's t-test.
Figure 4(A) Real-time quantitative reverse transcription polymerase chain reaction (PCR) (qRT-PCR) analysis of the differential abundance of selected bacterial taxa in stool samples of ileum and colon of Villin-Cre and COPS8-lox alleles expressing (COPS8fl/fl ) and COPS8 knockout (KO) (COPS8ΔIEC) mice. Error bars indicate the mean ± SD from three biological replicates per genotype. **P<0.01, NS- nonsignificant using Student's t-test. (Mice were treated by 2% dextran sodium sulfate (DSS) in drinking water for 7 days. (F) Representative hematoxylin and eosin (HE) stained sections of colon and cecum from COPS8fl/fl and COPS8ΔIEC mice after DSS-indcued colitis (day 9). Scale bar 100μm, representative data from five biological replicates per genotype. (G) Histopathologically scored sections of distal colon; cecum and colon length were analyzed from COPS8fl/fl and COPS8ΔIEC mice after DSS-indcued colitis (day 7). Data are represented as mean ± SEM from five biological replicates per genotype. **P<0.01 using Student's t-test. (Mice were treated by 2% dextran sodium sulfate (DSS) in drinking water for 7 days. (H) Cytokine levels (interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α)) in the colon collected on day 7 in colitis induced COPS8fl/fl and COPS8ΔIEC mice. Data are represented as mean ± SEM from five biological replicates per genotype. *P<0.05, **P<0.01, NS- non-significant using Student's t-test.Mice were treated by 2% dextran sodium sulfate (DSS) in drinking water for 7 days. (I) The frequency of CD11b+Ly6C+ and CD11b+Ly6G+ cells in colonic lamina propria (cLP) of COPS8fl/fl and or COPS8ΔIEC mice with DSS-induced colitis. The column graph represents percentage of Ly6C+ and Ly6G+ cells, presented as mean ± SEM from three biological replicates per genotype. *P<0.05, NS- non-significant using Student's t-test. (J) Representative FACS plots and percentage of intracellular staining of Forkhead box protein P3 (FOXP3), Interferon gamma (IFN-γ) and IL-17A in CD3+CD4+ T cells from colonic lamina propria of COPS8fl/fl or COPS8ΔIEC mice with DSS-induced colitis. The column graph represents percentage of Treg+ and ratio of Th1/Th17 cells, represented as mean ± SEM from three biological replicates per genotype. *P<0.05 using Student's t-test.
Figure 5(H) Flow cytometric analysis of CD45+ infiltrating immune cells in the lamina propria of the ileum. Data are represented as mean ± SEM from three biological replicates per genotype. **P<0.01, NS- non-significant using one-way ANOVA.(I) Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the expression of genes encoding inflammatory cytokines in the ileum. COPS8fl/fl and COPS8ΔIEC mice were either untreated (phosphate buffered saline (PBS)) or treated with the antibiotic cocktail (Abx) for 3 months. Data are mean ± SEM from three biological replicates. **P<0.01, NS- non-significant using one-way ANOVA.
Figure 6(B) Representative image showing changes in colon morphology and length following DSS-induced colitis with concurrent treatment with MBELNs COPS8fl/fl and COPS8ΔIEC mice. Representative data from seven biological replicates per genotype.Hematoxylin and eosin staining (HE) to show histological changes Scale bar 200μm(F) Expression of anti-microbial peptide (AMPs) in the colon of COPS8fl/fl and COPS8ΔIEC mice while being treated with MBELNs by oral administration. Data are mean ± SEM from three biological replicates per group. *P<0.05, **P<0.01, NS- non-significant using one-way ANOVA.(I-J) Transcriptional expression of nuclear factor kappa B (NF-κB) (I) and inhibitor of nuclear factor kappa-β kinase (IKK-β) (J) mRNA using Real-time quantitative reverse transcription Polymerase chain reaction (qRT-PCR) in colonic epithelial cells upon treatment with MBELNs in addition to DSS. Data are mean ± SEM from three biological replicates per group. **P<0.01, NS- non-significant using one-way ANOVA.
Figure 7(A) Western blots showing expression of AhR in MC38 cells induced by different fractions (protein, lipid and RNA) derived from Mulberry bark derived exosome-like nanoparticles (MBELNs). Data shown are from three biological replicates.(B) The effect of different size protein fractions of mulberry bark derived exosome-like nanoparticles (MBELNs) on the expression of aryl hydrocarbon receptor (AhR) demonstrated in Western blots. Data shown are from three biological replicates.(F) SPR experiment showing recombinant HSPA8 protein interaction with recombinant hAhR on a SPR chip. Data shown are from three biological replicates.
Figure 1TOPflash reporter assays in HEK293T cells stimulated with either control, Wnt3a, Wnt3a and RSPO1-4 conditioned media, or by transfection with the indicated constructs. Where indicated, cells were co-transfected with the indicated siRNAs.K, Representative co-immunoprecipitation experiments in HEK293T cells that were transfected with GFP-USP42 or the indicated GFP-tagged control, together with the stated Wnt signalling component (N = 3 independent experiments). IgG heavy chain is indicated with an asterisk. Dvl1 functions as a positive control that binds both ZNRF3 and Axin1.
Figure 2E,F, Co-immunoprecipitation experiments in HEK293T cells transfected with the constructs shown in (A). In (F) cells were co-transfected with DVL2 as indicated. Unspecific bands resulting from antibody cross-reaction are marked with asterisks. Representative blots of n ≥ 3 independent experiments are shown.
Figure 3A, Cell surface biotinylation assay performed in HEK293T cells transfected as indicated and treated with 10 μM MG132 to prevent proteasomal degradation. Consecutive immunoprecipitations were carried out as indicated in the scheme. The Avidin pulldown was carried under denaturing conditions (1st IP). The unrelated receptor CD147 was used as loading control for the avidin pull down, and CUL1 for the total input of cell lysate.F,G, Western blots of lysates from HEK293T cells transfected with the indicated constructs. The asterisk marks an unspecific band. Representative blots from at least three independent experiments are shown.
Figure 4D, TOPflash reporter assays in HCT116 cells upon knockdown of the indicated genes using single siRNAs. Cells were stimulated with control or Wnt3a conditioned medium. Data is displayed as mean ± SD, and show one representative of n = 3 independent experiments with three biological replicates.E,F, qPCR analysis of USP42, LGR5 and AXIN2 expression levels in HCT116 cells. Data are displayed as mean ± SD and show n = 4 (E) or n = 3 (F) independent experiments.
Figure 5E-H, Immunofluorescence microscopy showing endogenous E-cadherin levels in wt and WLS (Evi) KO HCT116 cells upon transfection with siUSP42 (E), or electroporation with eSpCas9 empty plasmid (non-edited) or eSpCas9-USP42 gRNAs(G). Scale bar = 20 μm. In (F,G), quantification of the fluorescent signal (Alexa-488) in n ≥ 4 biological replicates with n ≥ 15 cells per replicate are shown.I-L, Spheroid formation experiments with HCT116 cells upon transfection with siUSP42 (I-K) or electroporation with eSpCas9 empty plasmid (non-edited) or eSpCas9-USP42 gRNAs (L). Scale bar = 500 μm. Where indicated, cells were treated with 5 μM IWP-2 or 20 μM LGK-974 (PORCN inhibitors). The median size was quantified for n ≥ 13 spheroids from a representative experiment repeated independently n ≥ 3 is shown.
Figure 6A, Representative images of mouse small intestinal organoids generated with empty (Non-edited) or USP42gRNA containing CRISPR-concatemers, and cultured for 3 days in matrigel with the indicated growth factors, or the Wnt secretion inhibitor IWP-2. EN, EGF and noggin. Note that knockout of Usp42 (Usp42 gRNAs) rendered the organoids insensitive to RSPO1 withdrawal. Scale bar = 1 mm.C, qPCR analysis of Lgr5 and Axin2 expression levels in first passage mouse intestinal organoids cultured in EN + 5% RSPO1.