EMBO/SourceData huggingface dataset:
Figure 3(C-F') dmiR-1 binding site in 3'UTR-Mp region is required to negatively regulate Mp expression in vivo. Adult hearts from transgenic GFP-sensor lines carrying 3'UTRMp region with (UAS-GFP 3'UTRMp) or without dmiR-1 seed site (UAS-GFP ∆3'UTRMp). These lines were combined with the UAS-dmiR1 line to generate double transgenic lines and tested for GFP expression in non-targeted context (crossed with w1118) (C,E) or in heart-targeted context (crossed with heart specific driver Hand-Gal4) (D,F). When crossed with Hand-Gal4, GFP expression in Hand>GFP 3'UTRMp; dmiR1 line (carrying dmiR-1 seed site) (D') is attenuated in cardiac tube and pericardial cells compared to Hand>GFP ∆3'UTRMp; dmiR-1 line (lacking dmiR-1 seed site) (F'). Scale bar = 40 µm.(K,L) Graphs of Mp signal quantification in cardioblasts from adult flies aged one and five weeks for controls (UAS-mblRNAi, UAS-Bru3) and DM1 contexts (Hand>mblRNAi, Hand>Bru3) using CTCF method. For each genotype 9 hearts were analyzed and fluorescence intensity was measured in two regions from segment A3 and two regions from segment A4. Bars correspond to mean ± SD. Data information: p-value<0.05 considered statistically significant; (*) p-value = 0.033; (**) p-value = 0.021; (***) p-value = 0.0002; (****) p-value<0.0001. Non-parametric test Mann Whitney test was performed to compare control samples and samples of interest.
Figure 4(E-G) Box plots showing cardiac variables (diastolic (E) and systolic (F) diameters) and percent fractional shortening (G) for controls (Hand-Gal4 and UAS-Mp) and Mp overexpression (Hand>Mp) conditions at one and five weeks. Notice an increase in diastolic and systolic diameters with reduced cardiac contractility in Hand>Mp in comparison to controls. n = 20 hearts. Central band corresponds to median. Whiskers correspond to Min to Max. Box corresponds to interquartile range from 25th to 75th percentile. Data information: p-value<0.05 considered statistically significant. (*) p-value = 0.033, (**) p-value =0.021, (***) p-value =0.0002. Non-parametric Mann Whitney test was performed to compare control samples and samples of interest.
Figure 5(A-B') Adult heart of 1 week of age labeled for Mp (green) and actin (red) for control (UAS-Mp RNAi) (A,A') and mutant (Hand>Mp RNAi) contexts (B,B'). Data information: Scale bar = 20µm.(C) Fluorescence signal intensity quantification for Mp expression in cardioblasts in adult heart of 1 week for control (UAS-Mp RNAi) and mutant (Hand>Mp RNAi) contexts using CTCF method. For each genotype 9 hearts were analyzed and fluorescence intensity was measured in two regions from segment A3 and two regions from segment A4. Bars correspond to mean ± SD. Data information: p-value<0.05 considered statistically significant. (*) p-value = 0.033, (**) p-value =0.021, (***) p-value =0.0002. (****) p-value<0.0001. Non-parametric Mann Whitney test was performed to compare control samples and samples of interest.(D-F) Box plots showing cardiac size analyzes (diastolic (D) and systolic diameters (E)) and percent fractional shortening (F) performed by SOHA approach for controls (Hand-Gal4 and UAS-Mp RNAi) and mutant (Hand>Mp RNAi) contexts at 1 and 5 weeks of age, showing reduced size of cardiac tube in Hand>Mp RNAi context in comparison to the Hand-Gal4 control. n = 20 hearts. Central band corresponds to median. Whiskers correspond to Min to Max. Box corresponds to interquartile range from 25th to 75th percentile. Data information: p-value<0.05 considered statistically significant. (*) p-value = 0.033, (**) p-value =0.021, (***) p-value =0.0002. (****) p-value<0.0001. Non-parametric Mann Whitney test was performed to compare control samples and samples of interest.
Figure 6(A, B) (A) Col15A1 transcript levels tested by RT-qPCR and (B) Col15A1 protein levels tested by Western blot in cardiac samples from healthy donors and from DM1 patients with (DCM+) and without DCM (DCM-). Data information: p-value<0.05 considered statistically significant. (*) p-value = 0.033, (***) p-value =0.0002. (****) p-value<0.0001. Parametric t-test was performed to compare control samples and samples of interest in A, B(C) miR-1 levels tested by RT-qPCR in cardiac samples from healthy donors and from DM1 patients. Data information: p-value<0.05 considered statistically significant. (*) p-value = 0.033, (***) p-value =0.0002. (****) p-value<0.0001. Parametric t-test was performed to compare control samples and samples of interest in C.(D, F) Box plots showing cardiac variables (diastolic (D) and systolic (E) diameters and percent fractional shortening (F)) assessed by SOHA approach for controls (UAS-Bru3; Mp RNAi and UAS-UPRT; Bru3), Mp rescue (Hand>Bru3; Mp RANi) and DM1 context (Hand>UPRT; Bru3) at five weeks of age showing rescue of diastolic and systolic diameters in Hand>Bru3; Mp RNAi in comparison to controls (D,E) and rescue of cardiac contractility (F) n = 20 hearts. Central band corresponds to median. Whiskers correspond to Min to Max. Box corresponds to interquartile range from 25th to 75th percentile. Data information: p-value<0.05 considered statistically significant. (*) p-value = 0.033, (***) p-value =0.0002. (****) p-value<0.0001. Non-parametric Mann Whitney test was performed to compare control samples and samples of interest in D,E,F.
Figure 1A) Representative P2YR12 immunostaining (green) of microglia of wild type male mice showing morphological differences between control microglia (a) and ischemic microglia (b-d). Ischemic microglial cells show typical phagocytic pouches (arrows). Nuclei are labeled with TO-PRO-3 (blue). Image (d) is a magnification of the area marked with a square in (c). Scale bar a, b: 20 μm; c: 10 μm; d: 4 μm.
Figure 2C) PLIN2 protein expression in mouse brain tissue, as assessed by Western blotting. Values were obtained from the ipsilateral hemisphere 1h (n=3), 4h (n=3), 15h (n=4), 24h (n=5) and 96h (n=5) post-ischemia, and 15h (n=4), 24h (n=2), and 96h (n=2) after sham-operation. Values of sham mice were pooled together since they did not differ between time points. Samples of the contralateral hemisphere (Contra) of ischemic mice (1h, n=2; 24h, n=3; and 96h, n=3) were also evaluated. Ischemia increased PLIN2 expression at day 4 vs. the sham group (**p=0.0032, Kruskal-Wallis test and Dunn's multiple comparisons test). β-tubulin is the protein loading control. The 'Std' lane indicates the molecular weight standard. Quantification of band intensity where values are expressed as fold versus control (non-ischemic). Points correspond to independent male mice and group values are expressed as violin plot. Values were obtained 1h (n=3), 4h (n=3), 15h (n=4), 24h (n=5) and 96h (n=5) post-ischemia, and 15h (n=4), 24h (n=2), and 96h (n=2) after sham operation. Values of sham mice were pooled together since they did not differ between time points. Samples of the contralateral hemisphere (Contra) of ischemic mice (1h, n=2; 24h, n=3; and 96h, n=3) were also evaluated. Ischemia increased PLIN2 expression at day 4 (**p=0.0032, Kruskal-Wallis test and Dunn's multiple comparisons test vs. the sham group). Data are presented as violin plots with lines at the median and quartiles (dashed lines).D) Immunofluorescence with antibodies against PLIN2 (green) and Iba-1 (red) in brain tissue 1 day after induction of ischemia in female mice. Nuclei are stained with DAPI (blue). Control is the contralateral hemisphere. Scale bar: 10 μm.E) Transmission electron microscopy showing microglial cells in control or ischemic tissue one-day post-ischemia. Ischemia induces the formation of lipid droplets (LD) in microglia. LD are often seen near lysosomes (Lys). Insets in the lower panels are magnifications of the regions marked with a square. Scale bar 2 μm.F) Flow cytometry gates to identify Bodipy+ microglia for control and ischemic brain tissue. Gates were set based on fluorescence minus one (FMO) intensity values.G) Quantification of the percentage of CD11b+CD45low microglia containing lipid droplets as Bodipy+ microglia using flow cytometry in male mice deficient in Stat1 (Stat1-/-) (n=4) and corresponding Stat1+/+ mice (n=7) shows ischemia-induced increases in Stat1+/+ mice (** p=0.0086) but not in Stat1-/- mice (p=0.927) (Two-way ANOVA and Šídák's multiple comparisons test). Graph shows values of the non-ischemic (-) and ischemic (+) brain hemispheres of individual mice and the mean±SD.
Figure 5D) Quantification of flow cytometry shows that microglia renewal reduced the percentage of Bodipy+ microglia in old mice in sham (***p=0.0001) and ischemic (*p=0.0057) conditions (one-way Anova and Holm-Šídák's multiple comparisons test). For illustrative purposes the values of young mice are also shown (original old vs. young: ***p<0.001 in the sham group and **p=0.0026 in the ischemic group). Values show individual mice and bars show the mean±SD.E) Primary microglia cultures were obtained using CD11b+ magnetic beads from old male mice with or without microglia renewal (n=2 per group). Cells were exposed to red fluorescent pHrodo E. coli bioparticles and were stained with green fluorescent Bodipy and studied by confocal microscopy. Black and white images illustrate raw intensity data of Bodipy staining. The lower images are corresponding merged channels showing Bodipy in green, phagocytosed bioparticles in red, and the DAPI+ nuclei in blue. Repopulated microglia show less lipid droplets and the cytoplasm is replenished with phagocytosed bioparticles. The square regions of the images indicate areas magnified at the side of each image. Scale bar = 20 µm.F) Electron microscopy images representative of microglia of old male mice with original microglia (Old) and with renewed microglia (Old-Renewed) (n=3 per group). Images labelled (a-d) are higher magnifications of the areas marked with yellow squares in the images on the left. Lipid droplets are markes with arrows. Large lipid droplets are seen in microglia of old mice, whereas renewed microglia typically show no or small lipid droplets.
Figure 6C). The MRI lesion volume, as illustrated in the corresponding false color images below, showed no differences in infarct volume in mice with (n=16) or without (n=15) microglia renewal. Results correspond to day four post-ischemia. Representative MRI images obtained on day four post-ischemia using T2w MRI and day 14 using a Modified Driven Equilibrium Fourier Transform (MDEFT) MRI sequence (7.0 T BioSpec 70/30, Bruker BioSpin). Values show data for individual mice and/or the mean±SD.
Figure 1Anti-Flag Western Blot for transiently transfected FlagSLC25A51 variants and empty vector control in HeLa cells; HSP60, loading control.In vitro pull down of FlagSLC25A51 variants using cardiolipin-bound or control beads and detected with anti-Flag Western Blots.
Figure 2Free mitochondrial NAD+ levels measured using a ratiometric biosensor in HeLa cells expressing empty vector control (n = 25 biological replicates), wildtype FlagSLC25A51 (n = 25 biological replicates), and indicated mutants (n = 4 - 6 biological replicates). Measurements were taken at 24 hours post-transfection; the dashed line indicates the baseline defined by the empty vector control, and red denotes data equivalent to wildtype. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, ANOVA p <0.0001 with post-hoc Dunnett's test compared to empty vector control, *p < 0.05, **p < 0.01. (bottom) Protein expression from HeLa cells transiently transfected with empty vector control, wildtype FlagSLC25A51 and the indicated variants using anti-Flag western blot; HSP60, loading control.Quantitation of uptake of 32P-NAD+ after 1 hour by E. coli cells expressing wildtype SLC25A51 (amino acids 29-297) and the indicated mutants; wildtype activity and equivalent, red. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, n = 5 - 8 biological replicates, ANOVA p <0.0001 with post-hoc Dunnett's test compared to wildtype, ***p < 0.001.
Figure 3Free mitochondrial NAD+ levels measured using a ratiometric biosensor in HeLa cells co-expressing empty vector control (n = 8 biological replicates), wildtype FlagSLC25A51 (n = 8 biological replicates), and indicated mutants (n = 4 biological replicates). Measurements were taken at 24 hours post-transfection; the dashed line indicates the baseline defined by the empty vector control, and red denotes data equivalent to wildtype. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, ANOVA p < 0.0001 with post-hoc Dunnett's test compared to empty vector control ***p < 0.001. (bottom) Protein expression from HeLa cells transiently transfected with empty vector control, wildtype FlagSLC25A51 and the indicated variants using anti-Flag western blot; HSP60, loading control.Relative uptake of 32P-NAD+ after 1 hour by E. coli cells expressing wildtype SLC25A51 (amino acids 29-297) or the E132A mutant when competed by 100 μM (n = 3 biological replicates) and 250 μM (n = 4 biological replicates) unlabeled NAD+ (red) or NADH (black). Data is shown as floating bars with central line at mean and the box represents the min and max value, unpaired two-sided t-test *p < 0.05.Quantitation of 32P-NAD+ uptake after 1 hour in E. coli cells expressing wildtype SLC25A51 (amino acids 29-297) and indicated mutants; wildtype activity (n = 19 biological replicates) and equivalent, red. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, n = 5 - 8 biological replicates, ANOVA p <0.0001 with post-hoc Dunnett's test compared to wildtype ***p < 0.001.Free mitochondrial NAD+ levels measured using a ratiometric biosensor in HeLa cells expressing empty vector control (n = 27 biological replicates), wildtype FlagSLC25A51 (n = 27 biological replicates), and indicated mutants (n = 4 - 7 biological replicates). Measurements were taken at 24 hours post-transfection; the dashed line indicates the baseline defined by the empty vector control, and red denotes data equivalent to wildtype. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, ANOVA p < 0.0001, post-hoc Dunnett's test compared to empty vector control ***p < 0.001. (bottom) Protein expression from HeLa cells transiently transfected with empty vector control, wildtype FlagSLC25A51 and the indicated variants detected by anti-Flag western blot; HSP60, loading control.
Figure 4Free mitochondrial NAD+ levels measured using a ratiometric biosensor in HEK293 SLC25A51 KO cells expressing empty vector control, wildtype FlagSLC25A51, and indicated mutants. Measurements were taken at 48 hours post-transfection; the dashed line indicates the baseline defined by the empty vector control, and red denotes data equivalent to wildtype. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, n = 4 - 6 biological replicates, ANOVA p < 0.0001, post-hoc Dunnett's test compared to empty vector control **p < 0.01, ***p < 0.001. (bottom) Protein expression from HEK293 SLC25A51 KO cells transiently transfected with empty vector control, wildtype FlagSLC25A51 and the indicated variants detected with anti-Flag western blot; HSP60, loading control.
Figure 1(C, D) qRT-PCR analysis of Il2 and Ifng mRNA in CD4+ T cells from LN and OB mice (C) or from aged lean (AL, 17-month-old) and aged obese (AO, 17-month-old) mice (D), then stimulated with TCR (1/1 μg/ml) for 6 h. For AL and AO mice, mice were fed with a normal diet for 17 months, then divided into AL (≤ 35 g) and AO (≥ 45 g) mice based on body weight (n = 3 or 4, biological replicates). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.(J-L) BM12/SJL mice (recipient, R) were immunized with CD4+ T cells from LN and OB mice (donor, D), and then intraperitoneally injected once every day with 2-DG (200 mg/kg) or PBS for 3 weeks. Enzyme-linked immunosorbent assay (ELISA) of anti-dsDNA, anti-ssDNA, and anti-histone IgG or IgM in serum from the immunized mice at the indicated time (J). Immunofluorescent analysis of anti-nuclear antibody (ANA) in serum (top) and IgG deposition in the kidney (bottom) (n = 4 or 5, biological replicates). Scale bar: 50 μm. Data are presented as immunofluorescent images (K) and quantification of bar graphs (L). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.
Figure 2(F) qRT-PCR analysis of the transcriptional levels of indicated genes in fatty acid metabolism in CD4+ T cells ex vivo from LN and OB mice (n = 3 or 4, biological replicates). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.(L, M) ECAR measurements of glycolysis of naïve CD4+ T cell from LN and OB mice treated with vehicle or etomoxir (Eto, 150 μM) that stimulated with α-CD3/CD28 (TCR, 1/1 μg/ml) for 24 h (n = 3, biological replicates). The statistical results are presented as a bar graph (M). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.(S) BM12/SJL mice (recipient, R) were adoptively transferred with CD4+ T cells from WT and Cpt1a-KO lean or obese mice (donors, D) to induce inflammation. The anti-nuclear antibody (ANA) in serum (top, scale bar: 50 μm) and the kidney IgG deposition (bottom, scale bar: 50 μm) were visualized using immunofluorescence (n = 4 or 5, biological replicates). Data are presented as Immunofluorescent images (left) and quantification bar graphs (right). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.
Figure 3Immunoblot analysis of cytoplasmic and nuclear NF-AT levels (C), that activated by PMA/Ionomycin (P/I, 10/100 ng/ml) for the indicated time points.(R) Endogenous ubiquitination of Usp9x in WT and Goliath-deficient CD4+ T cells that were left unstimulated (-) or stimulated (+) with α-CD3/28 (5/5 μg/ml).(S) Co-IP analysis of the interactions between CaN and Usp9x or NFATc2 and CaN in WT and Goliath-KO CD4+ T cells that were left unstimulated (-) or stimulated (+) with α-CD3/28 (5/5 μg/ml).
Figure 4(A, B) Immunoblot analysis of cytoplasmic and nuclear NF-AT levels in WT and Goliath-KO naïve CD4+ T cells from lean (LN) or obese (OB) mice that were stimulated by α-CD3/28 (5/5 μg/ml) or PMA/ionomycin (P/I, 10/100 ng/ml).(C-F) ECAR measurements of glycolysis in WT and Goliath-deficient naïve CD4+ T cells treated with DMSO or fenofibrate (FF, 5 μM) (C, D), or isolated from LN or OB mice (E, F) that stimulated by α-CD3/28 (1/1 μg/ml) for 24 h (n = 3, biological replicates). The statistical results are presented as bar graphs (D, F). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.(J-L) BM12/SJL mice (recipient, R) were adoptively transferred with CD4+ T cells from WT and Goliath-KO lean or obese mice (donors, D) to induce inflammation. The anti-dsDNA, anti-ssDNA and anti-histone IgG or IgM in serum were examined by ELISA (J). ANA in serum (top, scale bar: 50 μm) and the kidney IgG deposition (bottom, scale bar: 50 μm) were visualized using immunofluorescence (n = 3, biological replicates). Data are presented as Immunofluorescent images (K) and quantification bar graphs (L). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.
Figure 5Immunofluorescence analysis of Goliath protein expression in CD4+ T cells from lean and obese mice as the indicated treatments. Data are presented as immunofluorescent images (A, and quantification bar graphs (B, Etomoxir (Eto) 150 μM for 6 h; Scale bar: 2 μM. Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, **p < 0.01, ***p < 0.001. Error bars represent SD.Immunofluorescence analysis of Goliath protein expression in CD4+ T cells from mice as the indicated treatments. Data are presented as immunofluorescent images C) Etomoxir (Eto), 150 μM for 6 h; MG132, 10 μM for the last 4 h. Scale bar: 2 μM.Immunofluorescence analysis of Goliath protein expression in CD4+ T cells from mice as the indicated treatments. quantification bar graphs D). Etomoxir (Eto), 150 μM for 6 h; MG132, 10 μM for the last 4 h. Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, **p < 0.01, ***p < 0.001. Error bars represent SD.(L) Endogenous K48-linked ubiquitination of HA-Goliath in GOLIATH-overexpressed Jurkat T cell with or without Cpt1a knockdown.(M) Endogenous ubiquitination of HA-Goliath with indicated treatment in GOLIATH-knockdown Jurkat T cell reconstituted with HA-tagged GOLIATH (WT) or Ring-truncated GOLIATH (ΔR).
Figure 6(E, F) Endogenous ubiquitination of Usp9x (E) and CaN (F) in primary CD4+ T cells treated with DMSO or Gonib32, then stimulated with (+) or without (-) PMA/ionomycin (P/I, 10/100 ng/ml).(G, H) Immunoblot analysis of the interaction of Usp9x and CaN, NF-AT and CaN (G), and NFATc1/2 in nuclear extract (NE) and cytoplasmic extract (CE) (H) in primary CD4+ T cells treated with Gonib32, then stimulated with (+) or without (-) P/I (10/100 ng/ml).ECAR measurements of glycolysis and proliferation analysis in lean and obese naïve CD4+ T cells treated with DMSO or Gonib32 (2 μM) upon α-CD3/CD28 (1/1 µg/ml) stimulation for 24 h (ECAR) (I, J) (n = 3, biological replicates). The statistical results are presented as a bar graph (J(M-O) BM12/SJL mice (recipient, R) were immunized with CD4+ T cells from lean and obese mice (donors, D), then were injected with Gonib32 (50 mg/kg) or vehicle control (5% DMSO + 95% saline) once a day for 3 weeks. Anti-dsDNA, anti-ssDNA, and anti-histone IgG or IgM in serum were measured using ELISA (M). Immunofluorescent analysis of anti-nuclear antibody in serum (top) and IgG deposition in the kidney (bottom) (n = 4 or 5, biological replicates). Data are presented as immunofluorescent images (N) and quantification bar graphs (O). All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.