EMBO/SourceData huggingface dataset:
Figure 5 A APOPT1HA was expressed in S6 and S2 immortalized fibroblasts as shown by the Western blot immunovisualization. The expression levels were tested at different days after transduction. The graph on the right shows the activities of CIV normalized to CS in three biological replicates per cell line. Data are presented as mean ± SEM (n = 3). The asterisks represent the significance levels calculated by two-way ANOVA with Tukey's multiple comparisons test: **P = 0.0030 (S6 naïve vs S6 APOPT1HA), *P = 0.0165 (S6 EV vs S6 APOPT1HA ) B APOPT1 GFP was expressed in S6 and S2 immortalized fibroblasts as shown by the Western blot immunovisualization. The graph on the right shows the activities of CIV normalized to CS in four biological replicates per cell line. Data are presented as mean ± SEM (n = 4). The asterisks represent the significance levels calculated by two-way ANOVA with Tukey's multiple comparisons test: **P = 0.0066 (S6 naïve vs S6 APOPT1GFP), ***P = 0.0006 (S6 EV vs S6 APOPT1GFP), *P = 0.0169 (S2 naïve vs S2 APOPT1GFP), ***P = 0.0008 (S2 EV vs S2 APOPT1GFP) C Western blot analysis of 1D-BNGE of mitoplasts extracted from the indicated cell lines. Fully assembled COX migrating at 200 kDa was immunovisualized with antibodies recognizing MT-CO1 and MT-CO2. The presence of the assembly intermediates 'MITRAC' and 'S3' was also detected (arrows)  D Western blot analysis of 2D-BNGE of mitoplasts extracted from the indicated cell lines. Fully assembled COX was immunovisualized with antibodies recognizing MT-CO1 and MT-CO2. The presence of the assembly intermediates 'MITRAC' and 'S3' was also detected (arrows). Anti-GFP immunodetection revealed the presence of the low molecular weight APOPT1GFP complex in the complemented cells expressing the tagged protein  E L-[35S]-Methionine pulse-chase labeling of mtDNA-encoded proteins. After a two-hour exposure with the radioactive label (pulse), cells were cultured in cold medium for the indicated chase times. The graphs show the densitometric quantification of the bands corresponding to MT-CO1 (left graph) and MT-CO2+MT-CO3 (right graph) normalized to the ATP6 band over the indicated time points. Graphs represent the values of three biological replicas for each cell line. Data are presented as mean ± SEM (n = 4 for controls, n = 2 for S2/S6/S2 APOPT1GFP/S6 APOPT1GFP). The asterisks represent the significance levels calculated by two-way ANOVA with Tukey's multiple comparisons test: MT-CO1 - **P = 0.0039 ( 3.5 hours, controls vs S6), *P = 0.0202 (3.5 hours, controls vs S2), *P = 0.0163 (5 hours, S6vs S6 APOPT1GFP), *P = 0.0118 (5 hours, controls vs S2), **P = 0.0011 (20 hours, controls vs S6), *P = 0.0343 (20 hours, S2 vs S2 APOPT1GFP), ***P = 0.0007 (20 hours, S6 vs S6 APOPT1GFP ), ***P = 0.0002 (20 hours, controls vs S2), *****P < 0.0001 (20 hours, controls vs S6), MT-CO2/MT-CO3 - *P = 0.0194 (3.5 hours, S6 vs S6 APOPTGFP), ***P = 0.0003 (3.5 hours, controls vs S6), *P = 0.0375 (5 hours, S6 vs S6 APOPT1GFP), *P = 0.0234 (20 hours, S2 vs S2 APOPT1GFP), ****P < 0.0001 (20 hours, S6 vs S6 APOPT1GFP), **P = 0.0013 (20 hours, controls vs S2), ****P < 0.0001 (20 hours, controls vs S6)
Figure 6 A Western blot analysis of SDS-PAGE of MGM132-treated 143B APOPT1HA cells. The graph represents the densitometric quantification of the signals for the precursor and mature protein  B The upper part of the panel (Input) shows Western blot analyses of APOPT1HA in the MGM132-treated cells. Note the appearance of higher molecular weight bands upon longer exposure in the samples treated with the proteasome inhibitor. The bottom part of the panel (Purified fractions) shows the analysis of fractions from the same cells immunoprecipitated with anti-HA. Note that the higher molecular weight species are cross-reacting with both anti-HA and anti-ubiquitin  C Western blot analysis of SDS-PAGE of total lysates from 143B cells overexpressing tagged APOPT1 (as indicated) and exposed to 100 µM H2O2, as illustrated by the scheme (H2O2 treatment), for the indicated times. The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point. The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to H2O2  D Western blot analysis of SDS-PAGE of total lysates from 143B cells overexpressing tagged APOPT1 (as indicated) and exposed to 5 µM MitoParaquat (MitoPQ), as illustrated by the scheme (MitoPQ treatment), for the indicated times. The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point. The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to MitoPQ
Figure 7 A Western blot analysis of SDS-PAGE of different mitochondrial proteins in total lysates from the indicated cell lines treated with 5 µM MitoPQ at the indicated times. UT: untreated cells. B Densitometric quantification of APOPT1GFP signal during the treatment in two biological replicas. C Densitometric quantification of MT-CO1 signal in the non-complemented APOPT1-less cells (S6 GFP) vs. the complemented cells (S6 APOPT1GFP). Three biological replicas (n=3) were carried out for each cell line. The signals in UT S6 APOPT1 were considered 100%. The levels of MT-CO1 were significantly lower in the S6 GFP patient cells after 20 hours of MitoPQ treatment compared to the untreated cells (*P = 0.0202, two-tailed unpaired Student's t-test). D Densitometric quantification of MT-CO2 signal in the non-complemented APOPT1-less cells (S6 GFP) vs. the complemented cells (S6 APOPT1GFP). Three biological replicas were carried out for each cell line. The signals in UT S6 APOPT1 were considered 100%
Figure 1A Western blots of eEF2 expression in different brain regions, GAPDH was used as a loading control. B eEF2 expression levels were quantified and represented as fold changes of that in the hippocampus. n=3 mice for each brain region. Data information: All data are shown as means ± s.e.m. All the t tests are two-tailed.C Immunofluorescent staining of eEF2 (green) and different nervous system cells (red: NeuN/GFAP/IBA1/OLIG2/CaMKⅡα/GAD65/67) in somatosensory cortex of adult mice brains. Arrows indicate highly colocalized cells. Scale bar, 10 μm.D, E Colocalization of eEF2 and each cellular marker was quantified by calculating the Manders' Colocalization Coefficients. 6 slices from 3 mice in each condition. (D) One-way ANOVA with Bonferroni's multiple comparisons post hoc, ****p<0.0001 (NeuN+/eEF2+ versus GFAP+/eEF2+), ****p<0.0001 (NeuN+/eEF2+ versus IBA1+/eEF2+), ****p<0.0001 (NeuN+/eEF2+ versus OLIG2+/eEF2+). (E) Unpaired t test, **p=0.0065 (CaMKⅡα+/eEF2+ versus GAD65/67+/eEF2+). Data information: All data are shown as means ± s.e.m. All the t tests are two-tailed.
Figure 2B Western blots of eEF2 in the PFC, the rest part of cortex, the hippocampus, and the striatum of Eef2 WT and HET mice.C Quantification analysis of (B). n=5 mice for cortex (without PFC), n=8 mice for PFC, n=9 mice for HP, n=3 mice for striatum. Unpaired t tests. Cortex (without PFC), p=0.9757; PFC, **p=0.0049; HP, p=0.1338; Striatum, p=0.7301. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.D Representative merged immunofluorescent staining images of eEF2 (green) and CaMKⅡα (red) in the mPFC of Eef2 WT and HET mice. Scale bar, 10 μm. E Quantification of mean fluorescent intensity of eEF2 in CaMKⅡα+ cells in WT and HET mPFC. n=918 cells from 3 WT mice, n=965 cells from 3 HET mice. Unpaired t test. **** p<0.0001. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.F Representative merged immunofluorescent staining images of eEF2 (green) and GAD65/67 (red) in the mPFC of Eef2 WT and HET mice. Scale bar, 10 μm. G Quantification of mean fluorescent intensity of eEF2 in GAD65/67+ cells in WT and HET mPFC. n=690 cells from 3 WT mice, n=686 cells from 3 HET mice. Mann-Whitney test. p=0.6757. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.H Western blots of de novo global protein synthesis in PFC slices of WT and HET mice, measured by SUnSET. α-tubulin was used as a loading control. I Quantification analysis of puromycin-labeled protein levels. WT, n=5 mice, HET, n=4 mice. Unpaired t tests. **p=0.0011. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.J Representative images of AHA incorporation (green) with CaMKⅡα+ staining (red) in mPFC of WT and HET mice. Arrows indicate CaMKⅡα+ cells. Scale bar, 20 μm. K Quantification of total AHA incorporation. 12 slices from 3 mice for each genotype. Unpaired t test. *p=0.0268. L Quantification of AHA incorporation in CaMKⅡα+ cells. WT, n=276 cells from 3 WT mice, n=293 cells from 3 HET mice. Mann-Whitney test. **** p<0.0001. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.
Figure 3G Representative images of Golgi‐stained dendrite spines in the mPFC from WT and HET mice. Scale bar, 5 μm. H Quantification of spine density per 10 μm of dendrites. n=18 dendrites from 3 WT mice; n=23 dendrites from 3 HET mice. Unpaired t test. ****p<0.0001. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.I Representative Western blots of expression levels of AMPAR and NMDAR subunits in the synaptosomal fraction of WT and HET PFC. α-tubulin was served as a loading control. J Quantification analysis of synaptosomal expression of different receptor subunits. Unpaired t test. GluA1, p=0.9346; GluA2, **p=0.0075; GluN1, p=0.6946; GluN2A, p=0.4091; GluN2B, p=0.0796. n.s., not significant. n=5 mice for each genotype. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.L Representative immunofluorescent images of eEF2 (magenta) and PLA signals (white) in lentivirus (LV)-scramble-EGFP or LV-Eef2 shRNA-EGFP infected primary cortical neurons (green, DIV 14). Scale bar, 10 μm. M Quantification of mean fluorescent intensity of eEF2 in EGFP+ cells in scramble and Eef2 shRNA groups. 64 cells for scramble group and 51 cells for Eef2 shRNA group from 3 independent experiments. Mann-Whitney test. ****p<0.0001. N Quantification of mean fluorescent intensity of PLA signals in EGFP+ cells in the two groups. The PLA signals represent newly synthesized GluA2. 64 cells for scramble group, 51 cells for Eef2 shRNA group from 3 independent experiments. Mann-Whitney test. ****p<0.0001. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.
Figure 7A PF-4778574 (PF) directly potentiates EPSC amplitude, dashed line represents baseline value of normalized EPSC amplitude. Bottom: representative EPSC traces from baseline (1), maximal potentiation (2), and recovery (3). B, C Quantifications of EPSC peak amplitude (B) and decay time (C) after PF (6 cells from 3 HET mice). (B) Wilcoxon signed-rank test, *p=0.0313 (PF v.s. baseline) and p=0.1600 (Wash out v.s. baseline); (C) Paired t test, ****p<0.0001 (PF v.s. baseline) and p=0.4423 (Wash out v.s. baseline). Data information: Data are shown as means ± s.e.m., dataD PF rescued AMPAR-EPSC in L5 neurons from HET mice. 25-30% increase at strongest stimulation (7 cells from 3 HET mice), paired t test, **p=0.0014. Bottom: representative traces of the effects of PF on AMPAR-EPSC evoked by different stimulus intensities. Data information: Data are shown as means ± s.e.m.E Representative heatmaps of the two social tasks (left, social approach; right, social novelty) in the Three-chamber test performed by Eef2 HET mice intraperitoneal administered with PF-4778574 or DMSO (vehicle control). E: empty cup; S1: stranger #1 contained cup; S2: stranger #2 contained cup. F Discrimination score of social approach performed by two groups of mice, calculated by the difference of time spent in sniffing S1 and E. Unpaired t test. p=0.7335. WT, n=9 mice; HET, n=8 mice. G Discrimination score of social novelty performed by two groups of mice, calculated by the difference of time spent in sniffing S2 and S1. Unpaired t test. *p=0.0492. WT, n=9 mice; HET, n=8 mice. Data information: Data F, G, are shown as interquartile range, with line across the box indicating median, whiskers show the highest and lowest values. n.s., not significant. All the t tests are two-tailed.I Representative heatmaps of the two social tasks (left, social approach; right, social novelty) performed by Eef2 HET mice with mPFC infusion of PF-4778574 or DMSO (vehicle control) in the Three-chamber test. E: empty cup; S1: stranger #1 contained cup; S2: stranger #2 contained cup. J Discrimination score of social approach performed by two groups of mice, calculated by the difference of time spent in sniffing S1 and E. Unpaired t test. p=0.3984. n=11 mice for each group. K Discrimination score of social novelty performed by two groups of mice, calculated by the difference of time spent in sniffing S2 and S1. Unpaired t test. *p=0.0489. n=11 mice for each group. Data information: Data of J and K are shown as interquartile range, with line across the box indicating median, whiskers show the highest and lowest values. n.s., not significant. All the t tests are two-tailed.
Figure 1Three independent samples (Exp 1 to 3) of EV secreted by labeled 3T3-F442A cells were analyzed by mass spectrometry (in duplicate injections, Inj1/2). The percentage of proteins bearing at least one peptide containing a heavy amino acid is indicated.
Figure 2Two human (SKMEL28 and 1205Lu) and a murine (B16BL6) melanoma cell lines were exposed, or not, to the indicated EV from primary murine adipocytes obtained from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD), then FAO was measured (n =5 for B16BL6, SKMEL28 HFD EV and 1205Lu ND EV and n=6 for other conditions).Volcano plot of mass spectrometry-based quantitative proteomics results showing relative abundance of proteins in primary murine adipocyte EV from obese mice (HFD), as compared to those from lean mice (ND). The dashed lines indicate cut-off values and points colored in grey indicate proteins that display non-significant fold-change by Welch t-test between both conditions (n=3 for ND, 4 for HFD). Proteins involved in FAO are indicated by yellow dots.Western blot analysis of the indicated FAO enzymes in the EV secreted by primary adipocytes from lean (ND) and obese (HFD) mice (top panel) and from human individuals with varying BMI (normal weight, NW; overweight, OW and obese, OB) (bottom panel). For each blot, extracts from 3 independent batches of murine samples or 3 independent individuals for human samples (1-3) are shown. Flotillin 1 (FLOT1) is used as a loading control.Western blot analysis of the indicated FAO enzymes in melanoma cells treated, or not, with EV from lean (ND) and obese (HFD) mice. Tubulin (TUB) is used as a loading control.RT-qPCR analysis of mRNAs for the indicated genes in 1205Lu cells treated or not with EV secreted by primary adipocytes from lean (ND) and obese (HFD) mice for 48 h. Results are expressed relative to the corresponding value for control cells (n=3).Analysis of FAO levels in 1205Lu cells exposed to 3T3-F442A EV and treated, or not, with cycloheximide (CHX) (n=5).
Figure 3Lipids were extracted from EV secreted by 3T3-F442A pre-adipocytes and differentiated 3T3-F442A adipocytes (respectively preAd-3T3-EV and Ad-3T3-EV), and FA content was measured (n=6).Indicated melanoma cells were incubated with EV from 3T3-F442A adipocytes previously loaded with BODIPY FL C16 (3T3-FL C16-EV) and, 24h later, cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy.Left panel, indicated cells were incubated with EV from 3T3-F442A adipocytes previously loaded with BODIPY FL C16 and immediately treated, or not, with Etomoxir for 24h. Then, cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy. Right panel, quantification of BODIPY FL C16 staining area per cell (n=3).Lipids were extracted from EV secreted by adipocytes from lean (ND) and obese (HFD) mice (n=5) (left panel) or from human adipose tissue samples from patients with varying BMI (right panel) and fatty acid content was measured (n=15).Indicated cells were exposed, or not, to adipocyte EV from primary adipocytes from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) for 24h. Then, cells were fixed, stained with BODIPY and counterstained with DAPI. Left panel, confocal microscopy observation. Right panel, quantification of BODIPY staining area per cell (n=5 for SKMEL28 and n=6 for 1205Lu).
Figure 4Transmission electron micrographs of 1205Lu cells exposed, or not, to 3T3-F442A EV (3T3-EV). Scale bar represents 1µm. A zoomed crop of the area with autophagic structures containing lipids (indicated by an arrow) is shown (scale bar: 0.5µm).1205Lu cells were incubated with EV from 3T3-F442A adipocytes, previously loaded with BODIPY FL C16 in the presence, or not, of Lalistat 2. Then, live cells were stained with the LysoTracker probe and observed by confocal microscopy. Arrows indicate colocalization.1205Lu were incubated with EV from 3T3-F442A adipocytes, previously loaded with BODIPY FL C16 and treated, or not, with Lalistat 2. Then, cells were fixed and counterstained with DAPI before observation by confocal microscopy. Quantification of BOBIPY FL C16 staining per cell is shown beside (n=4).1205Lu cells were exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Lalistat 2 (Lal). Cell migration was then evaluated in Boyden chamber assays (left panel; n=5) or cell motility was tracked by video microscopy (right panel; n=40 cells per group).1205Lu cells were exposed, or not, to adipocyte EV from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) with, or without, Lalistat 2 (Lal). Cells were then fixed, stained with BODIPY and counterstain with DAPI before observation by confocal microscopy. Quantification of BODIPY staining per cell is shown on the right (n=5).1205Lu cells were exposed, or not, to adipocyte EV from ND or HFD mice with, or without, Lalistat 2 (Lal). Cell motility was then tracked by video microscopy (n=40 cells per group).
Figure 51205Lu cells exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1, were stained with a MitoTracker probe, fixed and observed by confocal microscopy. Arrows indicate the presence of mitochondria in membrane protrusions. On the images to the right, the outline of cells is shown in dotted lines. Scale bars: 20µm. Right panel, quantification of the distance between mitochondria and nuclei (n=15).Western blot analysis of the indicated mitochondrial fission proteins in melanoma cells treated, or not, with EV from 3T3-F442A adipocytes (3T3-EV). Tubulin (TUB) is used as a loading control.1205Lu cells were exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1. Cell migration was then evaluated in Boyden chamber assays (left panel; n=6). Right panel, 3T3-F442A EV were added to 1205Lu cells, which were immediately treated, or not, with Mdivi-1. Cell motility was then tracked by video microscopy (n = 40 cells per group) (right panel).1205Lu cells were transfected, or not, with 2 different siRNA targeted against DRP1 (siDRP1#1 or 2) or an untargeted siRNA (siUT). Top, 48h after transfection, protein extracts were prepared and DRP1 expression was evaluated by western blot. Bottom, 36h post transfection, cells were exposed to 3T3-F442A EV (3T3-EV) and cell motility was tracked by video microscopy (n = 40 cells per group).1205Lu cells were exposed to adipocyte EV from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) and immediately treated, or not, with Mdivi-1. Cell motility was then tracked by video microscopy (n = 40 cells per group).
Figure 6Left panel, indicated melanoma cells exposed to 3T3-F442A EV were fixed, stained with BODIPY and Phalloidin before observation by confocal microscopy. Right panel, quantification of the percentage of cells presenting lipid droplets (LD) within membrane protrusions (n=6).Left panel, melanoma cells exposed to EV secreted by adipocytes from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) were fixed, stained with BODIPY and Phalloidin before observation by confocal microscopy. Right panel, quantification of the percentage of cells presenting lipid droplets (LD) within membrane protrusions (n=5 for SKMEL28 and n=6 for 1205Lu).1205Lu cells exposed to 3T3-F442A EV were stained with a MitoTracker probe. Then, cells were fixed and stained with BODIPY before observation by confocal microscopy. A zoomed crop of the area containing mitochondria and lipid droplets in a membrane protrusion is shown (scale bar: 5µm).Transmission electron microscope observations of 1205Lu cells exposed, or not, to 3T3-F442A EV. Mitochondria are colored in pink and lipid droplets are colored in yellow on images on the right.Number of lipid droplets (LD) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed, or not, to 3T3-F442A EV (3T3-EV) (n=28 for control and n=27 for 3T3-EV). Area of lipid droplets (LD) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed, or not, to 3T3-F442A EV (n=19 for control and n=25 for 3T3-EV).Left panel, 1205Lu melanoma cells were exposed, or not, to 3T3-F442A EV. Then, live cells were stained with LysoTracker and BODIPY probes and observed by confocal microscopy. Right panel, quantification of the percentage of cells presenting lysosome within membrane protrusions (n=3).
Figure 7TCGA data were analyzed to reveal the effect of high mRNA levels of key FAO enzymes on the overall survival of patients with melanoma. Statistical significance of survival was evaluated with a Log-rank (Mantel-Cox) Test (n=449, 450 and 459 for HADHA, HADHB and ECHS1 respectively).BODIPY staining of neutral lipid stores in the indicated melanoma cell lines, which present increasing degrees of aggressiveness (from left to right). DAPI was used to counterstain nuclei. Scale bar: 20µm.FAO in the indicated melanoma cell lines was measured. Bars and error bars represent means ± SEM (n=5); statistically significant by one-way ANOVA with post hoc Tukey's test, ***P < 0.001. If not indicated, non-significant.TCGA data were analyzed to reveal the effect of high mRNA levels of key actors in mitochondrial dynamics on the overall survival of patients with melanoma. Statistical significance of survival was evaluated with a Log-rank (Mantel-Cox) Test (n=459).
Figure 1(A) RT-PCR analysis for temporal expression of sinhcaf mRNA during embryogenesis and early larval developmental stages. NC indicates the no template control.(B) Time-course expression of sinhcaf revealed by whole-mount in situ hybridization. Scale bars, 100 μ(C) RT-PCR analysis for spatial expression of sinhcaf mRNA in adult tissues.(E) Relative mRNA level of sinhcaf in bud stage WT and MZsinhcaf -/- mutant embryos as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using unpaired two-tailed Student's t-test.(F) Fluorescence microscopy of 10hpf Tg(CMV:EGFP;CMV:sinhcaf) zebrafish embryos. Scale bar, 400 μm.(G) Relative mRNA level of sinhcaf in 10hpf or 24hpf WT and Tg(CMV:EGFP;CMV:sinhcaf) embryos as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using 2-way ANOVA followed by Holm-Sidak post-hoc test.(H) Percentage of male zebrafish in WT (n = 274), MZsinhcaf -/- (n = 166), Tg(CMV:EGFP;CMV:sinhcaf) (n = 158) and MZsinhcaf -/-;Tg(CMV:EGFP;CMV:sinhcaf) (n = 135) adult zebrafish. Data shown are mean ± SEM (n = the number of fish analyzed). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. The different letters (a, b, c) indicate a significant difference between the means, p<0.05.