EMBO/SourceData huggingface dataset:
Figure 8B Quantitative analysis of the body weight (g) of nondiabetic and diabetic mice transplanted with FI or PI+MVF from day -8 to day 28 (n = 7 each). Mean ± SD.C AUC of the body weight of nondiabetic and diabetic mice transplanted with FI or PI+MVF from day 0 to day 28 (n = 7 each). Mean ± SD.D Quantitative analysis of blood glucose level (mg/dL) of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls from day -8 to day 28 (n = 7 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. PI+MVF.E AUC of the blood glucose levels of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls from day 0 to day 28 (n = 7 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. PI+MVF.F Quantitative analysis of blood glucose level (mg/dL) according to the IPGTT of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls to the indicated time points (n = 5 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI.G AUC of IPGTT of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls (n = 5 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. PI+MVF.H Plasma insulin level (µU/mL) of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls at 180 min after IPGTT (n = 4 each). Mean ± SD.I Representative immunofluorescent stainings of insulin / CD31 in FI and PI+MVF on day 28 after islet transplantation. Scale bar: 50 µm.J Quantitative analysis of insulin- (β-cells), glucagon- (α-cells), somatostatin- (δ-cells) and CD31-positive (endothelial) cells within transplanted FI and PI+MVF on day 28 after islet transplantation. Data are expressed in % of total islet cells (n = 25). Mean ± SD.K Quantitative analysis of islet cells in transplanted FI and PI+MVF per HPF (n = 70 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. FI.
Figure 4(A) Extracellular D-serine concentrations in cortex were detected by enzyme-based microelectrode biosensors.(B, C) High performance liquid chromatography was performed to quantify total D-serine and L-serine contents in mouse brain of two genotypes. APP-KO mice were fed with D-serine over one month. One-way ANOVA was used for analysis followed by Bonferroni's multiple comparisons test and n>6 mice per group; * p<0.05, ** p<0.01 as compared to WT.
Figure 6(A) Quantification of total distance that mice traveled for 10 min.(B) The ratios of mice explored between novel and familiar objects. Lines in boxes indicate mean levels and whiskers represent the data from minimal to maximal range. The box extends from 25% to 75% of values. Wilcoxon signed-rank test was performed to compare data to the hypothetical value 1. N=9-12 mice per group; * p<0.05, ** p<0.01, NS - no significant difference.
Figure 1(D) Kaplan-Meier survival curves. Statistics: log rank Gehan Breslow Wilcoxon test, p < 0.0001, n=20 per group.
Figure 2(C) Kaplan-Meier survival curves of the 4 groups of mice shown in panel B. Statistics: Gehan-Breslow-Wilcoxon test.
Figure 3(B) Representative immunohistochemical staining for calreticulin in tumor masses of control (PBS) and BoxA-treated mice. Scale bar 50 μm. The distribution of calreticulin positive pixels describes the location of calreticulin (intracellular vs ecto-calreticulin). Statistics: Mann-Whitney test, n=4.(C) HMGB1 levels in the medium of AB1 cells treated with BoxA or 1 μM MTX for 24 hours. Statistics: One-way Anova with Dunnett's post-test; n=3. Data information: columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.(D) Ratio of phosphorylated eIF2α (p-eIF2α) over eIF2α in MM cells treated with increasing concentrations of BoxA or 1 μM MTX for 24 hours; β-actin is shown as loading control. Statistics: One-way Anova with Dunnett's post-test; n=2. Data information: columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.(G) Confocal immunofluorescence microscopy of cleaved caspase 3 (red) in MM cells treated for 24 hours with MTX (1 μM) or increasing concentrations of BoxA. Nuclei were stained with Hoechst (blue) and cytosol with phalloidin (green). Scale bar 50 μm.(I) Kaplan-Meier survival curve of mice injected with MM cells pretreated with PBS or BoxA shown in panel H (Log rank Gehan Breslow Wilcoxon test, p=0.59).
Figure 4(E) Representative immunofluorescence staining of CD47 (red) in unpermeabilized and permeabilized MM cells treated or not with 800 nM BoxA for 24 hours. Nuclei are counterstained with DAPI. Scale bar 50 μm.
Figure 5(A) Ecto-calreticulin exposure (expressed as MFI) in CT26 cells treated with 800 nM BoxA or 1 μM MTX for 2 and 4 hours. Error bars indicate standard deviation. Data were pooled from two different experiments. Data information: bars and error bars represent mean ± SD; statistics: One-way Anova with Dunnett's post-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.(I) Kaplan-Meier survival curves of mice shown in panel H. Statistics: Gehan-Breslow-Wilcoxon test.
Figure 6(A) Confocal images of Proximity Ligation Assays performed on CD47 and CXCR4, CD47 and RAGE, CD47 and TLR4. Representative images from one experiment out of 3 performed are shown. MM cells were incubated overnight with 400 nM BoxA or PBS. Nuclei are in blue (DAPI), phalloidin is in green. Red dots represent physical contact of CD47 with CXCR4 or RAGE or TLR4. Scale bar, 20 μm. The intensity of red signal in PLA was quantified as described in Materials and Methods in individual cells (n as indicated). Insets are enlargements of the y axis. Mean and SD are indicated. Statistics: Kolmogorov-Smirnov test.
Figure 7(C) Surface CD47 on MM cells after incubation for 24 hours with the indicated concentrations of CXCL12. The experiment shown is representative of 2 performed, in biological triplicate. Data information: bars and error bars represent mean ± SD; statistics: one-way Anova plus Dunnett's post-test. In all panels, *p<0.05, **p<0.01, ****p<0.0001.
Figure 1Impact of MB on cytotoxicity of OT-1 CTLs against EG7 or EG7-L1. Splenocytes from OT-1 mice in culture were stimulated with 10 nM of OVA257-264 (SIINFEKL) for 3 days to generate mature CTLs. CTLs were incubated with CFSE labeled EG7-L1 cells in the presence of MB at indicated concentrations. Cytotoxicity was determined by flow cytometry analysis. Data are representative of three independent experiments. (effector to target ratio = 5:1, unpaired t-test). EG7-L1: EG7 overexpressing PD-L1.   Statistical results of (B).   ELISA measured of LDH release to assess the cytotoxicity of OT-I CTLs on EG7-L1 in the presence of MB. Anti-PD1 antibody (aPD-1) served as positive control.   Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001.Impact of MB on cytotoxicity of PD-1-/- (PD-1KO) OT-1 CTLs against EG7 or PD-L1 expressing EG7 stable cell line. Data are representative of three independent experiments. (unpaired t-test). Statistical results of (E). Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001.Cytotoxicity of OT-I CTLs against B16-F10-OVA cells (designated B16-OVA) in the presence of 1 μΜ of MB or DMSO. Cytotoxicity was determined by the relative area unoccupied by cells examined under microscope (scale bar = 50 μm). Data are representative of three independent experiments, and were analyzed by unpaired t-test.   Statistical results of (G).   Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001.
Figure 4Schematic of the xenograft mouse model for MB treatment. C57BL/6J mice were inoculated with EG7-L1 cells (2 × 106 cells, s.c.) on the right flank on day 1, followed by injection (2 × 106 cells, i.v.) of CD45.1+ CTL on day 3 and day 6 respectively. The mice were randomized into 3 groups (n = 5) and treated with vehicle, MB (20 mg/kg/day, i.g.), aPD1 (10 mg/kg, every other day, i.p.) served as positive control. Tumor size was measured through calipering every other day. EG7-L1: EG7 overexpressing PD-L1.   Tumor growth curve for (A) was shown as mean ±s.e.m.   Representative image of the tumor on day 10 mentioned in (A).   Tumor weight on day 10 (n=5) mentioned in (A).   Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.Immunohistochemical analysis of cell proliferation marker ki-67 and CD8 in tumor nodules on day 10 (n=5) Statistical results of (E). Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.Expression of Perforin, GZMB and IL-2 by CD8+ CD45.1+ TILs were analyzed by flow cytometry.   Statistical results of (G).   Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.Schematic of EGFR L858R mice treated with MB.   Tumor burdens recorded through Computed tomography (CT) scanning for EGFR L858R mice (n=5, lung section = 3). Mice were treated with MB (20 mg/kg/day, i.g.) for 1 week, followed by imaged with CT again to record tumor burden.   Statistical results of (J).   Haematoxylin and Eosin staining of lung section of EGFR L858R mice (n = 5, lung section = 3) in (J).   Statistical results of (L).   Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Figure 5Effect of MB on proliferation of human CD8+ T cells. CFSE labeled human peripheral blood mononuclear cells (PBMC) were pre-incubated with DMSO, MB, 25 μg/ml of pembrolizumab or 20 μg/ml of Nivolumab for 1 h and then seeded in the 96-well plate precoated with 10 μg/ml of aCD3/aCD28 in the presence of 10 μg/ml human PD-L1 Protein. Cell proliferation was analyzed through FACS analysis of dilution of CFSE. Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001.Cytokine and cytolytic granule production in CD8+ T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96-well precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD-L1 Protein. Cytokine and cytolytic granule production were determined by intracellular flow cytometry by gating on CD8+ populations. Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001.Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP-luc cells were stimulated with Raji-L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25μg/ml of pembrolizumab (Pem) or 20μg/ml of nivolumab (Niv) for 6 hours. Luciferase activity was measured by luminometer. Data are representative of three independent experiments, and were analyzed by unpaired t-test. JP-luc: Jurkat cell harboring NFAT-luciferase transgene and overexpressing PD-1; Raji-L1: Raji overexpressing PD-L1. Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001.
Figure 1Curated heatmaps of top 76 up and 72 down regulated genes. Highlighted genes were chosen based on biological interest. The coloured bar represents the expression level from low (green) to high (red).
Figure 2Real-time qPCR analysis of fetal organoids cultured for one month showing decrease in relative expression of indicated neonatal markers A Ass1, B Blimp-1, C FcRn and D Lct and increase of the adult markers E Sis, F Treh and G Arg2 (n=3 individual wells from single organoid culture (see material and methods); experiment was repeated in four to eight independent organoid cultures with similar results).Enzyme activity assay of fetal organoids for H Lactase, I sucrase, J maltase, K trehalase and L arginase. Activity is given in µM glucose/µg protein·min-1 (experiment was generated from single organoid culture (see material and methods) and repeated in 3 independent organoid cultures with similar results).
Figure 3Relative expression detected by Real-time qPCR in fetal organoids ( ) and adult organoids ( ). Neonatal markers A Ass1, B Blimp-1, C FcRn and D Lct in fetal organoids cultured for one month compare to levels adult organoids. Mature markers E Sis, F Treh and G Arg2 increase in fetal organoids to adult levels (n=3 individual wells from single organoid culture (see material and methods); experiment was repeated in four independent organoid cultures with similar results).Enzyme activity assay of fetal ( ) and adult ( ) organoids. H Lactase activity is higher in fetal organoids decreasing to adult levels at day 28, while I sucrase, J maltase, K trehalase and L arginase increase to adult levels. Activity is given in µM glucose/µg protein·min-1 (n=3 independent organoid cultures).
Figure 4Maturation is accelerated in dexamethasone treated fetal organoids ( ) compared to control ( ), as revealed by real-time qPCR. Decrease in relative expression of neonatal marker A Blimp-1 and increase in expression of adult marker B Sis occur earlier in dexamethasone treated organoidsDexamethasone treatment accelerates increase in activity of sucrase maltase reaching adult organoid level ( ) at day 13 of the culture. Activity is given in µM glucose/µg protein·min-1 (n = 3 independent organoid cultures).Maturation is accelerated in dexamethasone treated fetal organoids ( ) compared to control ( ), as revealed by real-time qPCR. expression of adult marker E Treh and F Arg2 remains unchanged (n=3 individual wells from single organoid cultureDexamethasone treatment accelerates increase in activity of G trehalase and H arginase reaching adult organoid level ( ) at day 13 of the culture. Activity is given in µM glucose/µg protein·min-1 (n = 3 independent organoid cultures).
Figure 5Microscopic images of fetal organoids at day 3, 13, 20 and 30 of culture. Scale bars: 500 µm.Percentage of fetal spheroids decreases during culture (n=3 individual wells from single organoid culture (see material and methods); experiment was repeated in ten independent organoid cultures with similar results).Immmunohistochemistry of fetal organoids for C Ass1 D Sis and E Arg2. Quantification of 3 slides per staining, 7 to 10 organoids / spheroids per slide. Scale bars: 50µm.
Figure 1(C) All eight subunits of the CCT chaperonin complex were enriched in U2OS-A3A samples. Proteins detected by MS are displayed by fold enrichment (log2) from U2OS-A3A cells compared to parental cells (x-axis), and by protein abundance within the U2OS-A3A samples (y-axis). Significantly enriched proteins (p<0.05 by one-tailed t-test, n=3) are denoted in dark blue. Components of the CCT complex are highlighted and labeled.
Figure 2(A) A3A co-precipitates with CCT complex subunits. U2OS and HepaRG cells with dox-inducible A3A-HA transgenes were treated with dox prior to immunoprecipitation of HA. HA immunoprecipitates were analyzed by immunoblot with antibodies for HA and subunits of the CCT complex.(C) A3A interacts with CCT in leukemia cells. HA immunoprecipitation in hematopoietic cell lines with inducible A3A-HA was analyzed by immunoblot. HA, CCT1, and CCT5 antibodies were used to evaluate co-precipitation of CCT subunits with A3A.
Figure 3(A) CCT co-precipitates with A3A but not A3B or A3G. 293T cells were transfected with empty vector (V), A3A-HA, A3B-HA, or A3G-HA. CCT IP was performed from transfected cells and evaluated by immunoblot analysis using antibodies for CCT5 and HA.(B) A3A, but not other APOBEC3 enzymes, interacts with CCT complex subunits. After transfection of 293T cells with HA-tagged APOBEC3 expression plasmids, IP was performed with anti-HA antibody and analyzed by immunoblot using antibodies to HA and CCT subunits. Arrows mark location of each APOBEC3 protein on HA immunoblot.(F) CCT co-precipitates with endogenous A3A. Immunoprecipitation of the CCT complex in BICR6 cells was analyzed by immunoblot using antibodies to endogenous A3A and CCT1. Chemiluminescent exposure times described as short and long are indicated beside the blot. IgG antibody was used as a control throughout.
Figure 4(A) Knockdown of one CCT complex member results in depletion of other CCT subunits. Following siCCT4 or siCCT7 transfection, cell lysates were evaluated by immunoblot for expression of additional CCT subunits by endogenous antibodies for CCT1, CCT4, CCT5, and CCT7. GAPDH was used as a loading control.(B) Decreased cell viability upon CCT depletion and A3A expression. CCT4 (left) and CCT7 (right) subunits were depleted by siRNA transfection in U2OS cells with dox-inducible A3A transgenes. Immunoblot analysis of cell lysates showed decreased expression of targeted CCT subunit, but no alteration in A3A expression. Scr indicates non-targeted siRNA, used as control. Ku-86 is a loading control. Following siRNA transfection to deplete CCT subunits, U2OS-A3A cells were treated with dox. Viability was determined by colorimetric change after addition of a water-soluble tetrazolium salt. Percent viability was normalized to untreated controls. Statistical analysis was performed using a paired two-tailed t-test, n=3 biological replicates; error bars, SEM.(D) Viability of cells with catalytically inactive A3A. U2OS-A3A-C106S cells were induced with dox to express the catalytically inactive A3A mutant (C106S), depleted of CCT4 by siRNA, or combination. Cells were subsequently evaluated by live-dead staining assessed by FACS. Bar chart shows quantitation of FACS results averaged over three biological replicates. Statistical analysis was performed using a paired two-tailed t-test, n=3; error bars, SEM. Immunoblot analysis of cell lysates showed decreased expression of CCT4 after siRNA targeting. Ku-86 is a loading control.(E) Increased DNA damage signaling upon CCT depletion and A3A expression. Depletion of CCT7 in K562-A3A cells was achieved by siRNA targeting. Expression of A3A was induced by dox treatment. Intracellular staining with anti-γH2AX antibody was analyzed by flow cytometry. Bar chart shows quantification of FACS results obtained over three biological replicates. Statistical analysis was performed using a two-tailed t-test, n=3; error bars, SEM. Immunoblot analysis of cell lysates showed decreased expression of CCT7 after siRNA targeting and A3A expression upon dox treatment. Ku-86 is a loading control. **p-value <0.01, ***p-value <0.01, ****p-value <0.0001, n.s. non-significant.
Figure 5(C) Quantification of APOBEC3 mutational signatures among three tumor types that have previously been associated with high APOBEC3 activity. Breast, bladder, and cervical cancer samples from the TCGA database were divided into CCT-mutated and non-mutated genomes, as above. The number of mutations contributing to SBS 2 and SBS 13 in each tumor type was quantified and displayed as an average. Comparison by Wilcoxon rank sum test yielded p-values denoted.
Figure 1Blue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. Immunodetection of the indicated proteins after BNGEBlue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. Immunodetection of the indicated proteins after BNGEBlue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. (C) in-gel activity for CI and (D) CIV (shown is a representative gel from two technical and two biological replicates).BNGE of whole-body zebrafish digitonin-solubilized mitochondria of scaf1Δ1/Δ1 (-/-) and its respective scaf1+/+ counterpart. Immunodetection of the indicated proteinsBNGE of whole-body zebrafish digitonin-solubilized mitochondria of scaf1Δ1/Δ1 (-/-) and its respective scaf1+/+ counterpart. (G) in-gel activity of CI and (H) CIV (representative gel from two technical and three biological replicates).I, 2D BNGE/SDS electrophoresis: 1st dimension with digitonin (Dig) and 2nd dimension with SDS, followed by immunoblotting with the indicated antibodies to identify the proteins detected by the commercial anti-SCAF1 antibody. Asterisks indicate missing bands in scaf1Δ1/Δ1.
Figure 2A, Quantitative data-independent scanning (DiS) mass spectrometry protein profiles for CI, CIII and CIV. Vertical numbers indicate the BNGE gel slices. Left and right profiles correspond to scaf1+/+ and scaf1Δ1/Δ1 animals, respectively. Red heatmap corresponds to the E-score from two proteotypic Scaf1-derived tryptic peptides spanning sequences ascribed to the CIII interacting site (in green) and to the CIV interacting site (in yellow). Thick blue line, marked with an asterisk, indicates the putative proteolytic site in Scaf1.
Figure 3A, B, Representative images from scaf1+/+ and scaf1-/- (A) female and (B) male adult zebrafish.c-f, Size of scaf1Δ1/ Δ1 and scaf1Δ2/ Δ2 (scaf1-/-) fish in comparison with their respective scaf1+/+ lines, (C) length and (E) weight of females (Δ1 +/+ n=10, Δ1 -/- n=12, Δ2 +/+ n=24, Δ2 -/- n=18); (D) length and (F) weight of males (Δ1 +/+ n=16, Δ1 -/- n=13, Δ2 +/+ n=13, Δ2 -/- n=23).G-K, Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (G, I) Adipose tissue area per total section area (average of 3 sections/biological replicate) and (H, J) adipocyte size (average of 20-30 adipocytes of ventral adipose tissue per biological replicate) of females (G, H) (Δ1 n=8, Δ2 n=8, same number of animals for homozygous mutants and controls) and males (I, J)(Δ1 n=8, Δ2 n=7, same number of animals for homozygous mutants and controls). K, Representative images of ventral fat deposits in females (dotted lines).L-N, Effect of Scaf1 loss of function on female fertility. (L) Number of eggs per clutch (Δ1 +/+ n=12, Δ1 -/- n=13, Δ2 +/+ n=13, Δ2 -/- n=10). (M) Quantification of mature ovary follicles per ovary section (average of three sections/biological replicate) (Δ1 n=5, Δ2 n=8; same number of animals for homozygous scaf1+/+ and scaf1-/-). (N) Representative images of H&E-stained ovaries. Dotted lines delineate adipose tissue.