EMBO/SourceData huggingface dataset:
Figure 4(A-B) PLA of CV-bound ATPIF1 produces a red fluorescence signal where anti-ATPIF1 and anti-ATP5A1 antibodies are localized at a distance less than 10nm of each other. Anti-TOMM20 (green) was used to reveal mitochondrial architecture. (A) Representative high-resolution Airyscan confocal micrographs showing control fibroblasts treated with EPI for 24hrs. Maximum intensity projection is shown. Scale bars: 20 µm and 5 µm in zoomed image. Note that PLA reveals the spatial distribution of ATPIF1 binding along the mitochondrial length. (B) Analysis of ATPIF1-CV complexes occurrence quantified as PLA dots per µm3of mitochondria. Effect of EPI is shown relative to untreated. (n=2 from >50 replicates per n). In all cases, for each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.(E) mtATP content measured in control and human mitochondrial disease fibroblasts treated for 30min with increasing concentrations of EPI as indicated. Analysis shows the mean intensity of mtATP probe in MTG area normalized per % of correspondent untreated cells (n=3). In all cases, for each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.
Figure 5PLA of CV-bound ATPIF1 (red). Anti-TOMM20 (green) was used to reveal mitochondrial architecture. (A) Representative confocal micrographs showing fibroblasts treated with EPI for 24hrs. Maximum intensity projection is shown. Scale bars: 20 µm and 5 µm zoomed images. (B) Analysis of ATPIF1-CV complexes occurrence quantified as PLA dots per µm3of mitochondria in patient fibroblasts normalized in % of Ctrl untreated (n=2 from >50 replicates per n). In all cases, data represent average ± SEM. For each biological replicate, technical replicates were averaged. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.PLA of CV-bound ATPIF1 (red). Anti-TOMM20 (green) was used to reveal mitochondrial architecture. (C) Effect of EPI on ATPIF1-CV complexes occurrence quantified as PLA dots per µm3 under the indicated conditions. Values are shown as relative to control untreated. In all cases, data represent average ± SEM. For each biological replicate, technical replicates were averaged. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.(E-G) Effect of EPI on CIII-deficient (BCS1L) cells stable-expressing ATPIF1WT or ATPIF1H49K. (E) CV ATP hydrolytic capacity measured by the acidification rate and normalized maximal respiration. Maximal respiration was determined using the OCR channel (Max OCR on SR). Effect of EPI is shown as % of control cells, untreated with EPI. Cells were treated for 24hrs with 50nM EPI (n≥4). (F) mtATP content measured as fluorescence intensity per mitochondria area and shown as % of control untreated (n=5). Mitochondrial area was quantified using MTG staining. Cells were treated for 30min. (G) Total cellular ATP content shown as % of untreated cells. Cells were treated for 24hrs (n≥4). In all cases, data represent average ± SEM. For each biological replicate, technical replicates were averaged. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.
Figure 6D2.mdx mice, harboring the mdx mutation in a DBA/2J genetic background (D2.mdx) were subjected to exercise induced gastrocnemius injury by stimulating muscle contraction (eccentric injury). Levels of CV, ATPIF1 and maximal CV ATP hydrolytic capacity were measured. (A) Representative western blot image (left) and quantification (right) showing CV (ATP5A1) and CII (SDHA) levels in gastrocnemius homogenate in WT and mdx mice after 24hrs of eccentric injury (inj). Vinculin was used as loading control. Note that only in mdx mice eccentric injury results in reduced CV content. n=4. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.D2.mdx mice, harboring the mdx mutation in a DBA/2J genetic background (D2.mdx) were subjected to exercise induced gastrocnemius injury by stimulating muscle contraction (eccentric injury). Levels of CV, ATPIF1 and maximal CV ATP hydrolytic capacity were measured. (C) Analysis of levels of the cleaved OPA1 in gastrocnemius homogenate in WT and mdx mice 24hrs after eccentric injury. n=4. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.
Figure 7D2.mdx mice were treated with EPI orally twice daily, with doses ranging from 0.5 to 15 mg/kg/day. After 13 days of treatment, mice were subjected to exercise induced gastrocnemius injury by stimulating muscle contraction and the effect of EPI on muscle force were measured. (A) Plantarflexor force quantification after eccentric injury. n=8. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. unpaired t-tests (A, shows statistical differences depicted by p-value.D2.mdx mice were treated with EPI orally After 13 days of treatment, mice were subjected to exercise induced gastrocnemius injury by stimulating muscle contraction and OPA1 cleavage were measured. (E) Levels of cleaved OPA1 in gastrocnemius homogenate from mdx mice subjected to the indicated combinations of injury and EPI treatment. n=8. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two-way ANOVA followed by Šídák's multiple comparisons testD2.mdx mice were treated with EPI (G) Detection and quantification of myofiber membrane integrity or damage following injury using Evans Blue dye (EBD) in gastrocnemius muscle from vehicle and EPI treated mdx mice. Damaged fiber membranes allow dye entry that appears as red color. Yellow asterisks indicate damage fibers (n≥4). unpaired t-tests G) shows statistical differences depicted by p-value.
Figure 1(A) Pictures of plants treated with BLE. Plants were grown vertically on 1/2 MS medium with or without 4 μM BLE for 9 days . Scale bar = 1 cm. (B) The relative root length of the indicated plants. The data are represented as means ± SD (n = 10 plants) relative to the values of Col-0 under the control condition. The statistical significance was determined using two-way ANOVA analysis. ****, P < 0.0001. The experiments were repeated three times with similar results.(H and I) Representative GUS staining images of cotyledons (H) and quantification of the relative HR efficiency (I). The reporter line and trigger line in either ddrm4-1 or Col-0 background were crossed and the F1 seedlings were used for GUS staining analysis. Scale bar = 1 mm. The number of blue sectors was scored. The data are presented as means ± SD (n > 132 cotyledons) relative to the values of Col-0. The statistical significance was determined using two-tailed Students' t-test. ****, P < 0.0001.
Figure 2(A-F) (A, C, and E) Representative images of CFP-PAF1 expressed in N. benthamiana leaves (A), Arabidopsis protoplasts (C), and transgenic Arabidopsis (E). The N. benthamiana leaves and the transgenic seedlings were treated with 40 μM BLE for 4 h before imaging. The Arabidopsis protoplasts were treated with 20 μM BLE for 2 h before imaging. Scale bars in (A and C) = 2.5 μm. Bars in (E) = 10 μm. (B, D, and F) The percentage of cells with CFP-PAF1 foci in N. benthamiana leaves (B), Arabidopsis protoplasts (D), and transgenic Arabidopsis (F). The data are represented as means ± SEM (n = 3 biological replicates). For each sample, more than 35 cells were scored. The statistical significance was determined using two-tailed Students' t-test.(K) eChIP-qPCR assays. The immunoprecipitated DNA and the input DNA were subjected to qPCR analysis. The ratios of eChIP and input are shown. UBQ5 serves as a negative control. The positions of the PCR fragment of 0.5 kb-4.5 kb were shown in (I). The data are represented as means ± SD (n = 3 technical replicates) relative to the values of Mock. The statistical significance was determined using two-tailed Students' t-test. ns, not significant; **, P < 0.01; ***, P < 0.001. The experiments were repeated three times with similar results.
Figure 3(A and B) Yeast two-hybrid assays. PAF1 was fused with the activation domain (AD). SMC5 and SMC6B were fused with the DNA binding domain (BD). DDO, double dropout (SD/-Trp/-Leu) medium. QDO, quadruple dropout (SD/-Trp/-Leu/-His/-Ade) medium.(G and H) In vitro pull-down assays. The Dextrin beads coupled with MBP, MBP-SMC5, or MBP-SMC6B were incubated with His-PAF1, respectively. After washing, the beads were subjected to western blotting analysis using anti-His or anti-MBP antibodies.
Figure 4The PAF1 foci are dependent on SMC5 and SMC6B. (A) CFP-PAF1 was transfected alone or co-transfected with the RNAi construct of SMC5 (SMC5-Ri) into Col-0 protoplasts. (C) CFP-PAF1 was transfected into the protoplasts of Col-0 or smc6b, respectively. The protoplasts were treated with 20 μM BLE for 2 h before imaging. The percentage of cells with foci. The data were represented as means ± SEM (n = 3 biological replicates). For each sample, at least 32 cells were scored. The statistical significance was determined using two-way ANOVA analysis. **, P < 0.01; ***, P < 0.001.(G and H) eChIP-qPCR assays. (G) The SMC5-Ri construct was transfected into the protoplasts of SSDIS/CFP-PAF1 transgenic plants. (H) The 35S:CFP-PAF1 construct was transfected into the protoplasts of SSDIS/smc6b or SSDIS/Col-0 transgenic plants. The protoplasts were treated with 100 μM DEX for 4 h to induce DSBs. The eChIP assays were carried out using an anti-GFP antibody. The immunoprecipitated DNA (eChIP) and the input DNA were subjected to qPCR analysis. The ratios of eChIP and input are shown. UBQ5 serves as a negative control. The data are represented as means ± SD (n = 3 technical replicates). The statistical significance was determined using two-tailed Students' t-test. ns, not significant; ****, P < 0.0001.
Figure 5(C and D) BiFC assays. The proteins were fused to either the C- or N-terminal half of YFP (cYFP or nYFP) and were transiently expressed in N. benthamiana. The YFP fluoresce detected by confocal microscopy indicates interaction. Scale bars = 10 μm.(E and F) CoIP assays. The fusion proteins were co-expressed in N. benthamiana leaves. The proteins precipitated by anti-GFP beads were subjected to western blotting analysis using anti-FLAG or anti-GFP antibodies.(G and H) In vitro pull-down assays. The Dextrin beads coupled with MBP or MBP-UBC2 were incubated with His-CDC73 or His-VIP5. After washing, the beads were subjected to western blotting analysis using anti-His or anti-MBP antibodies.(I and J) The UBC2 foci are dependent on VIP5. UBC2-mCherry and/or VIP5-YFP were transfected into the vip5 protoplasts. The protoplasts were treated with 20 μM BLE for 2 h before imaging. Scale bars = 2.5 μm. The data in (J) were represented as means ± SEM (n = 3 biological replicates). For each sample, at least 30 cells were scored. The statistical significance was determined using two-way ANOVA analysis. ****, P < 0.0001. All experiments were repeated at least three times with similar results.
Figure 6(C and D) In vitro pull-down assay. The Dextrin beads coupled with MBP, MBP-HUB1, or MBP-HUB2 were incubated with His-UBC2. After washing, the beads were subjected to western blotting analysis using anti-His or anti-MBP antibodies. The experiments were repeated three times with similar results.(E-J) The HUB1/2 foci are dependent on UBC2. VIP5-CFP, HUB1/2-YFP, and/or UBC2-mCherry were transfected into the ubc1 ubc2-c1 protoplasts. The protoplasts were treated with 20 μM BLE for 2 h before imaging. Bars = 2.5 μm. The data in (F, G, I, and J) were represented as means ± SEM (n = 3 biological replicates). For each sample, at least 60 cells were scored. The statistical significance was determined using two-way ANOVA analysis. ns, not significant; **, P < 0.01.
Figure 7(A, C, E, and G) Pictures of plants treated with BLE. Plants were grown vertically on 1/2 MS medium with or without 6 μM BLE (A) or 4 μM BLE (C, E, and G) for 8 days. Scale bar = 1 cm. (B, D, F, and H) The relative root length of the indicated plants. The data are represented as means ± SD (n = 10 plants) relative to the values of Col-0 under the control condition. The statistical significance was determined using two-way ANOVA analysis. ns, not significant; ***, P < 0.001; ****, P < 0.0001. The experiments were repeated three times with similar results.(I and J) eChIP-qPCR assays. (I) The SSDIS/Col-0 transgenic Arabidopsis was mock-treated or treated with 100 μM DEX for 4 h. (J) The SSDIS/Col-0 and SSDIS/ddrm4-1 transgenic Arabidopsis were treated with 100 μM DEX for 4 h. The eChIP assays were carried out using an anti-H2Bub antibody. The immunoprecipitated DNA (eChIP) and the input DNA were subjected to qPCR analysis. The ratios of eChIP and input are shown. UBQ5 serves as a negative control. The data are represented as means ± SD (n = 3 technical replicates). (I) The data are relative to the values of Mock. (J) The data are relative to the values of UBQ5 in Col-0. The statistical significance was determined using two-tailed Students' t-test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. The experiments were repeated at least twice with similar results.
Figure 1(B) Indicated cell lines grown for 72 hours in complete medium or medium deficient in arginine and cell density recorded (x105/mL). 3 independent experiments are shown with mean and SEM. The dotted line shows seeding density (10,000 cells/100µL). Data information: For statistical analysis, an ordinary one-way ANOVA with Dunnett's correction for multiple comparisons was performed(C) Indicated cell lines grown for 72 hours in complete medium or medium deficient in arginine and viability measured. 3 independent experiments are shown with mean and SEM. The live cell fractions (Annexin V-, 7-AAD-) were used for statistical analysis. Data information: For statistical analysis, an ordinary one-way ANOVA with Dunnett's correction for multiple comparisons was performed(D) Western blotting was used to visualise ASS1 protein levels in untreated or imatinib treated (2µM, 48 hours) CML CD34+ samples, normal CD34+ samples and indicated cell lines following 16 hours arginine starvation.(E) ASS1 expression from the MILE study. The dotted line revers for average of Healthy Bone Marrow. Data information: For statistical analysis, an ordinary one-way ANOVA with Dunnett's correction for multiple comparisons was performed a Kruskal-Wallis test was performed on E with **** referring to P<.0001 in E.
Figure 2(A) CTV analysis of CML CD34+ patient samples after 72 hours arginine starvation (n=3). Data are shown with mean and SEM. Data information: For statistical analysis, multiple unpaired tests with Benjamini, Krieger, and Yekutieli Benjamini two-stage step up correction was performed out on log-transformed data in A(B) Viability data from CML CD34+ patient samples recorded after 72 hours starvation of indicated amino acid. Data are shown are from 3 biological replicates with mean and SEM plotted. Mean and SEM are shown. Data information for B-E Mann-Whitney tests were performed.(D) CFCs from CML CD34+ patient samples generated after 72 hours starvation of indicated amino acid. The upper panel shows representative image of colonies, counts are plotted in the lower panel. Data are shown are from 4 (-arginine) or two (-ornithine or -citrulline) biological replicates. Mean and SEM are shown. (E) CFCs from Normal CD34+ samples generated after 72 hours starvation of indicated amino acid. Upper panel shows representative image of colonies, counts are plotted in lower panel. Data are shown are from 3 biological replicates. Mean and SEM are shown. Data information: Mann-Whitney tests were performed.
Figure 3(B) Indicated cell lines (Control or knock-down (sg-2 or sg-3)) grown for 24 hours in absence or presence of BCT-100 (1000ng/mL) and ASS1 protein levels measured. Data information: For statistical analysis, ordinary one-way ANOVA with Tukey's correction for multiple comparisons was performed(C) Indicated cell lines (Control or knock-down (sg-2 or sg-3)) grown for 72 hours in absence or presence of BCT-100 (1000ng/mL) and viability measured. 3 independent experiments are shown with mean and SD. Live cells (Annexin V-, 7-AAD-) were analysed. Data information: For statistical analysis, ordinary one-way ANOVA with Tukey's correction for multiple comparisons was performed(E) Viability data from CML CD34+ patient samples (5 biological replicates) recorded after 72 hours of treatment as in D, Mean and SEM are shown. Data information: For statistical analysis, ordinary one-way ANOVA with Tukey's correction for multiple comparisons was performed(G) CFCs from CML CD34+ samples are shown. Cells were treated as in D before seeding for CFCs. The left panel shows representative image of colonies, counts are plotted in the right panel. Data are shown are from 7 biological replicates. Mean and SEM are shown. Data information: For statistical analysis, ordinary one-way ANOVA with Tukey's correction for multiple comparisons was performed
Figure 4(B) Volcano plot showing differentially expressed (DE) genes between CML CD34+ vehicle and BCT-100 treated cells, red denotes q-value <0.1. Data information: , DESEQ2 and GSEA were used as described in methods.
Figure 5Absolute concentration of indicated amino acid in serum is shown. Dashed line denotes lowest linear point from standard curve. Data information: Metabonalyst was used to conduct both two-way ANOVA and Multivariate Empirical Bayes Analysis (MEBA) on R-Log transformed data in A and B.(F) Representative data showing CD34+CD38- population from each treatment group.(G) The percentage of CD34+CD38- cells (from CD34+) is shown. Biological replicate data from all mice, average and SD are plotted. Vehicle: n=8 mice, Imatinib: n=8 mice, BCT-100: n=6 mice, Combo: n=7 mice. Data information A Kruskal-Wallis test was used to analyse data in G(H) The absolute number of CD34+CD38- cells is shown. Biological replicate data from all mice, average and SD are plotted. Vehicle: n=8 mice, Imatinib: n=8 mice, BCT-100: n=6 mice, Combo: n=7 mice. Data information A Kruskal-Wallis test was used to analyse data in H.
Figure 2A. Representative western blot of hTERT-RPE1 cells transfected for 72 hours with siRNA against indicated targets or non-targeting (NT1) prior to harvesting and cell lysis.B. Quantification of BNIP3, NIX and HIF1α protein levels for data represented in A. Individual datapoint from n=5 (BNIP3, NIX) or 4 (HIF1α) independent colour coded experiments are shown. Shown is the fold change normalised to NT1 control. Error bars show standard deviation. One way ANOVA and Dunnett's multiple comparison test **P<0.01, ****P<0.0001.
Figure 3A. Representative western blot of two matched pairs of control (Ctrl) and FBXL4 KO pool RPE1-Cas9i-mt-mKeima cells, generated with two distinct sgRNAs. B. Quantification of data represented in A. Individual datapoints from n=3 independent colour coded experiments are shown. Error bars show standard deviation of the fold change in BNIP3 and NIX in each KO pool normalised to their matched control pools; one-way ANOVA and Tukey's multiple comparison's test, **P<0.01, ****P<0.0001.D. Representative images of control or FBXL4 KO pool RPE1-Cas9i-mt-mKeima cells. "Magenta only" puncta (Ex. 561) correspond to acidic mitolysosomes. Scale bar 20 µm. E. Graphs show the quantification of mitolysosomes (mt-mkeima "magenta only" puncta) per cell. A minimum of 45 cells were analysed per condition in each experiment. Individual data from two (for control 1 (Ctrl 1) and knockout 1 (KO1)) or three (control 2 (Ctrl 2) and knockout 2 (KO2)) are shown using different colours for each independent experiment. Mean and standard deviation are indicated. Bottom graph shows the individual data points. One-way ANOVA with Tukey's multiple comparison's test, *P<0.05.G. Quantitative RT-PCR reactions of BNIP3 and NIX (normalised to Actin) were performed with cDNA derived from control and FBXL4 KO RPE1-Cas9i-mt-mKeima cells. Two primer pairs were used per target indicated by symbol shape. Three independent colour-coded experiments are shown. Error bars show standard deviation of the fold change of BNIP3 and NIX mRNA in each KO pool normalised to their matched control pools.
Figure 4A and B. hTERT-RPE1 cells were co-transfected with Flag (V) or FBXL4-Flag (FL4) and either GFP (Vect), GFP-BNIP3 or GFP-NIX. Lysates were subjected to immunoprecipitation (IP) with either GFP-nanobody coupled beads (A) or Flag-antibody coupled agarose beads (B). IPs were probed alongside the input samples for Flag and GFP.D. hTERT-RPE1 cells were transfected with empty vector (V), FBXL4-Flag (WT) or FBXL4- R482W-Flag (RW). Lysates were subjected to IP with Flag-antibody coupled agarose beads and probed for SKP1 and Flag. Note that FBXL4-Flag presents with an as yet uncharacterized higher molecular weight band (indicated by a blue arrowhead) E. Quantitation of SKP1 protein co-immunoprecipitated with FBXL4-R482W-Flag relative to FBXL4-Flag (WT). Data from two independent experiments are shown, normalised to the amount of SKP1 in the input and the FBXL4-Flag in the IP.F and G. Representative images of Control (Ctrl) and FBXL4 KO RPE1-Cas9i-mt-mKeima cells transfected with FBXL4-Flag wild-type (WT) or FBXL4-Flag [R482W] for 24 hours, then fixed and stained for either BNIP3 or NIX. Scale bar 20 µm. Quantitation of the frequency of high BNIP3 and high NIX phenotypes observed in F and G. A minimum of 32 Flag-positive (Transfected, indicated by an asterisk in the images) and Flag-negative cells were analysed per condition in each experiment. Shown are the mean and individual datapoints of two colour-coded experiments.
Figure 5Control (Ctrl) or FBXL4 KO RPE1-Cas9i-mt-mkeima cells were treated for 72 hours with either non-targeting (NT1), BNIP3 (B3), NIX or HIF1α (HIF) siRNA. A. Representative images of the mt-mKeima reporter in Control (Ctrl2) or FBXL4 KO2 RPE1-Cas9i-mt-mkeima. "Magenta only" puncta (Ex. 561) correspond to acidic mitolysosomes. Scale bar: 20 µm.C. Representative western blot verifying the knockdown efficiency for samples shown in B.
Figure 6C. Representative western blot of lysates prepared from hTERT-RPE1 cells transfected with siRNA targeting BNIP3 (B3), NIX or both for 72 hours. Cells were treated with either DMSO or MLN4924 (1 µM) for the last 24 hours of transfection. Untransfected cells were treated alongside transfected ones with DMSO, antimycin and oligomycin (AO; 1 and 10 µM respectively), or MLN4924. D. Quantification of fold change in protein levels calculated from data represented in C for three colour-coded independent experiments; One way ANOVA and Dunnett's multiple comparison test *P<0.05, ****P<0.0001.E. Representative western blot of hTERT-RPE1 cells treated with either DMSO or MLN4924 (1 µM) for the indicated time points.G. Quantification of fold change in protein levels calculated from data represented in F for six colour-coded independent experiments. Error bars show standard deviation; One way ANOVA and Tukey's multiple comparison test **P<0.01, ****P<0.0001.
Figure 7A. Representative western blot of lysates from control or FBXL4 KO RPE1-Cas9i-mt-mKeima cells treated with MLN4924 (1 µM) for 6 hours. B. Representative western blot of lysates from control or FBXL4 KO RPE1-Cas9i-mt-mKeima cells pre-treated without or with MLN4924 (1 µM) for 6 hours followed by a cycloheximide (CHX) chase (100 µg/ml).C and D. Quantification of protein levels calculated from data represented in B for three colour-coded independent experiments. Error bars show standard deviation.F. TUBES-pulldown of ubiquitylated NIX. FBXL4 KO RPE1-Cas9i-mt-mkeima cells and matched control cells were treated as in E and lysates subjected to a TUBES pulldown.