EMBO/SourceData huggingface dataset:
Figure 4C) the quantitation (C) of intracellular free [D3]-m6rA-related nucleotides in WT and Adal-/- mES cells. Total free [D3]-m6rA = Free [D3]-m6rA + [D3]-m6rAMP + [D3]-m6rADP + [D3]-m6rATP. Error bars = s.d., three independent biological replicates. *The standard was not labeled with heavy stable isotope.E) quantitation (E) of intracellular free [D3]-6mdA-related nucleotides in WT and Adal-/- mES cells. The cells were treated with [D3]-methionine for 7 days. Total free [D3]-6mdA = Free [D3]-6mdA + [D3]-6mdAMP + [D3]-6mdADP + [D3]-6mdATP, Error bars = s.d., three independent biological replicates. *The standard was not labeled with heavy stable isotope. ND, not detected.
Figure 5The western blot analysis of flag-tagged gene overexpression in Adal-/- mES cells (A) and NIH3T3 cells (B) overexpressing ADK, APRT, AK1 or NDPK gene.(C) The quantification of genomic 6mdA in [15N5]-rA-traced Adal-/- mES cells overexpressing flag-tagged ADK, APRT, AK1 or NDPK gene for 7 days. (D) The quantification of genomic 6mdA in NIH3T3 cells overexpressing ADK, APRT, AK1 or NDPK gene. [15N5]-rA treatment began at 24 h after plasmid transfection and lasted for 72 hours. Two technical replicates.The knockdown of Ak1 leads to a significant decreased i6mdA in NIH3T3 cells. western blot (F) were applied to determine the knockdown of Ak1 in NIH3T3 cells. Two independent biological replicates. (G) The quantitation of misincorporated 6mdA in Ak1-knockdown cells. [15N5]-rA treatment began at 24 h after siRNA transfection and lasted for 48 hours. Error bars = s.d., three technical replicates.
Figure 1(B) Western blot analysis of the generated isogenic cell lines described in A after inducing GFP-TDP-43 expression for 48 h showing the tightness of the doxycycline (DOX)-modulated expression system. Note also the different protein levels of the expressed variants. endTDP-43: endogenous TDP-43.(C) Representative images of widefield fluorescence microscopy of the isogenic cell lines depicted in B. GFP brightness is adjusted in each condition for optimal visualization of GFP-TDP-43 localization. Original intensity values are represented in the right column using grayscale. Cell nuclei are stained with DAPI. Scale bar: 20 µm.(D) GFP-TDP-43 expression was induced with DOX for 24 h before cycloheximide (CHX) treatment for the indicated times and western blot analysis. (E) Quantification of the GFP signal from D. N=3 independent experiments. Two-way ANOVA with Tukey's multiple comparisons post hoc test.
Figure 2(B) Representative maximum intensity Z-projections from confocal fluorescence imaging of human neurons transduced with TDP-43-HA variants and immunolabeled for the HA tag and the neuron-specific marker MAP2. Scale bar: 10 µm. Cell nuclei are stained with DAPI(D) GFP-TDP-43 expression was induced with doxycycline (DOX) for 4 h before nucleocytoplasmic fractionation and subsequent analysis of GFP-TDP-43 and endogenous TDP-43 (endTDP-43) levels by western blot. T: total lysate, C: cytoplasmic fraction, N: nuclear fraction. (E) Quantification of the GFP signal from D. Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Tukey's multiple comparisons post hoc test. Cyt: cytoplasm, Nuc: nucleus.(F) HEK293 cells were treated with ActD to inhibit transcription or subjected to protein-protein cross-linking with DSG followed by nucleocytoplasmic fractionation and western blot analysis. T: total lysate, C: cytoplasmic fraction, N: nuclear fraction, H3: histone H3. (G) Quantification of endTDP-43 signal from F. N=3 independent experiments. Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Dunnett's multiple comparisons post hoc test.(H) Representative images of widefield fluorescence microscopy of HEK293 cells treated with ActD for 4 h and immunolabeled for TDP-43. Scale bar: 20 µm. (I) Quantification of nucleocytoplasmic levels of endTDP-43 in the immunocytochemistry images shown in H. N=3 independent experiments. Unpaired two-tailed t-test. Cell nuclei are stained with DAPI(J) Representative images of confocal fluorescence microscopy of human neural cultures treated with ActD and ivermectin (IVM) and immunolabeled for TDP-43 and the neuron-specific marker MAP2. Scale bar: 20 µm. (K) Quantification of nucleocytoplasmic levels of endTDP-43 in the immunocytochemistry images shown in J. Kruskal-Wallis test with Dunn's multiple comparisons post hoc test. N=23-48 fields corresponding to a total of 351-569 neurons per condition. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Graph bars represent mean ± SD. Violin plots show mean and quartiles. Cell nuclei are stained with DAPI
Figure 3(A) Representative maximum intensity Z-projections from confocal fluorescence imaging (thickness of ~10 µm, in steps of 0.21 µm) of HEK293 cells after mock or 1,6-hexanediol (1,6-HD) treatment for 15 min stained for endogenous TDP-43 (endTDP-43). Scale bar: 5 µm. (B) Quantification of three-dimensional (3D) reconstructions from the images of the conditions shown in A depicting the number of nuclear condensates per cell. N=14-23 cells. Unpaired two-tailed t-test.(C) Fluorescence microscopy images of 10 μM purified full-length TDP-43 and its oligomerization-deficient counterpart showing different abilities to undergo LLPS and its disruption by 1,6-HD treatment for 10 min. Scale bar: 10 μm. (D) Quantification of the number of condensates in the conditions shown in C per 4000 µm2 field. N=10 independent experiments. Kruskal-Wallis test with Dunn's multiple comparisons post hoc test.(H) GFP-TDP-43 expression was induced with DOX for 4 h before crosslinking protein-protein interactions with DSG and subsequent analysis by western blot. *, ** and *** indicate GFP-TDP-43 monomers, dimers and trimers, respectively. o, oo, ooo and oooo indicate endTDP-43 monomers, dimers, trimers and tetramers. ­­­(I) Quantification of GFP-TDP-43 dimer/monomer ratio based on the GFP signal from H. N=3 independent experiments. Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Tukey's multiple comparisons post hoc test. * p<0.05, ** p<0.01, **** p<0.0001. Graph bars represent mean ± SD.
Figure 4(A) HEK293 cells were treated with actinomycin D (ActD) for 4 h to inhibit transcription before treatment with the protein-protein cross-linker DSG and western blot analysis. o, oo and oooo indicate endogenous TDP-43 (endTDP-43) monomers, dimers and trimers, respectively. (B) Quantification of the endTDP-43 signal from A. N=3 independent experiments. Paired two-tailed t-test.(C) Representative image of confocal fluorescence microscopy of HEK293 cells and neurons treated with 5 μg/ml ActD for 4h. Scale bar: 5 µm. (D) Single-plane quantification of the number of nuclear condensates per neuron in the conditions described in C. N=25-26 cells. Mann-Whitney U test.(F) PLA using a monoclonal anti-GFP antibody reveals the localization of GFP-TDP-43 dimers in the isogenic cell lines upon protein expression with doxycycline (DOX) for 48 h. Note the absence of dimers in the oligomerization-deficient variants (6M and 6M&RRMm). Scale bar: 5 µm.(G) Quantification of the nuclear PLA signal shown in F correlated to the protein expression levels of the different TDP-43 variants, measured as the mean GFP fluorescence. N=11-13 cells.
Figure 5(A) Representative confocal microscopy images of HEK293 cells probed for endogenous TDP-43 (endTDP-43) and different subnuclear compartment markers. Lower panel shows the three-dimensional (3D) reconstruction of the endTDP-43 condensates and the indicated nuclear body obtained from confocal Z-stacks. Scale bar: 5 µm. (B) Quantification of the 3D reconstructions shown in A depicting the percentage of nuclear TDP-43 condensates colocalizing with markers of subnuclear compartments. N=14-38 cells. (C) Quantification of the 3D reconstructions shown in A depicting the percentage of each of the analyzed subnuclear compartments that colocalize with endTDP-43. N=14-38 cells.(D) Representative confocal microscopy images of human neurons showing the presence of endTDP-43 in Cajal bodies. The neuron-specific marker MAP2 is shown in cyan. Scale bar: 20 µm (inset: 5 µm).(E) Representative confocal microscopy images of the isogenic HEK293 lines expressing the different GFP-TDP-43 variants for 24 h and stained for the Cajal body marker coilin. Scale bar: 5 µm. (F) Representative confocal microscopy images of the isogenic HEK293 lines expressing the different GFP-TDP-43 variants for 24 h and hybridized with a fluorescent NEAT1 probe to mark the paraspeckles. Scale bar: 5 µm.
Figure 6(A) Volcano plots showing alternative splicing (AS) events upon expression of GFP-TDP-43 variants for 48 h.(E) Western blot analysis of the isogenic HEK293 after GFP-TDP-43 expression for 48 h showing that only the WT variant regulates endTDP-43 levels. (F) Quantification of the endTDP-43 signal from E. N=4 independent experiments. Two-way ANOVA with Tukey's multiple comparisons post hoc test.(G) Tripartite GFP complementation assay involving the co-transfection of a pair of N-terminally T10- and T11-tagged TDP-43 constructs in HeLa cells subjected to arsenite stress for 30 min and incubated with recombinant GFP1-9 after fixation to label T10- and T11-TDP-43 dimers. TriFC: trimolecular fluorescence complementation. Scale bar: 10 µm. (H) Quantification of the trimolecular fluorescence complementation (triFC) signal of GFP in the TIA-1-marked SGs from the images shown in G. N=3 independent experiments. Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Tukey's multiple comparisons post hoc test.(I) Expression of GFP-TDP-43 mutNLS variants was induced with doxycycline (DOX) for 4 h before crosslinking protein-protein interactions with DSG and subsequent analysis by western blot. * and ** indicate GFP-TDP-43 monomers and dimers, respectively. (J) Quantification of the GFP signal from I. N=3 independent experiments. Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Tukey's multiple comparisons post hoc test.(K) After expression of GFP-TDP-43 mutNLS variants for 48 h, the isogenic lines were treated with DSG to cross-link protein-protein interactions before performing nucleocytoplasmic fractionation and western blot analysis. * and ** indicate GFP-TDP-43 monomers and dimers, respectively.
Figure 7(A) Representative confocal microscopy images of the isogenic GFP-TDP-43 lines after 48 h of expression, treated with the proteasome inhibitor MG132 for the last 24 h and stained for lamin B to mark the nuclear envelope. Note the different localization of TDP-43 inclusions in the oligomerization- (6M and 6M&RRMm) versus RNA binding-deficient (RRMm) variants. Scale bar: 20 µm (5 µm for inset). (B) Quantification of the localization of GFP-TDP-43 inclusions after MG132 treatment for the different variants in the isogenic HEK293 lines as shown in A. Represented values are averages from N=3 independent experiments, with N=189-497 cells quantified per condition and replicate.(C) Representative maximum intensity Z-projections from confocal fluorescence imaging (thickness of 4 µm, in steps of 1 µm) of human neurons transduced with TDP-43-HA variants and treated overnight with the proteasome inhibitor MG132. Scale bar: 10 µm. (D) Quantification of the differentially localized TDP-43-HA inclusions in human neurons as described in C. Represented values correspond to the quantification of N=85-97 neurons from two independent experiments. Nuclei are stained with DAPI The neuron-specific marker MAP2 is shown in magenta or cyan(E) Representative maximum intensity Z-projections from confocal fluorescence imaging (thickness of 4 µm, in steps of 1 µm) of the same experimental conditions as shown in A, with the addition of a DMSO control for MG132 treatment. GFP-TDP-43-expressing cells were double immunolabeled for TDP-43 phosphorylated at the S403/404 and S409/410 epitope. Note the absence of phospho-S409/410 immunopositivity in all conditions and positivity for phospho-S403/404 in cytoplasmic inclusions and a subset of nuclear GFP-TDP-43 RRMm inclusions (appointed by arrowheads). Scale bar: 10 µm. Nuclei are stained with DAPI(F-G) Representative maximum intensity Z-projections from confocal fluorescence imaging (thickness of 4 µm, in steps of 1 µm) of the same experimental conditions as shown in C, with the addition of a DMSO control. TDP-43-HA expressing human neurons were stained for either phospho-S403/404 (F) or phospho-S409/410 (G). Scale bar: 10 µm. Note the re-localization of the phospho-S403/404 signal from neuronal nuclei to inclusions (F) and the presence of phospho-S409/410 signal in only a subset of aggregate-bearing neurons (G). Nuclei are stained with DAPI The neuron-specific marker MAP2 is shown in magenta or cyan
Figure 8(B) Representative fluorescent confocal microscopy images of FRAP experiments of the GFP-TDP-43 aggregates formed upon MG132 treatment as described in Figure 7A. FRAP was performed in the areas highlighted in magenta. Numbers in images indicate the experimental time point in seconds ('') of MG132 treatment. Scale bar: 5 µm. Bottom panel: Measured GFP intensity values are expressed as a fraction of the average pre-bleach fluorescence levels.(D) Representative confocal microscopy images of the isogenic GFP-TDP-43 lines at the endpoint (24 h) of MG132 treatment of the experimental conditions described in Figure 7A and stained for vimentin. Scale bar: 5 µm. Cell nuclei are visualized with DAPI(E) Representative confocal microscopy images of the isogenic GFP-TDP-43 lines at the endpoint (24 h) of MG132 treatment of the experimental conditions described in Figure 7A and stained for p62. Cell nuclei are visualized with DAPI
Figure 1(B) Immunoblot analysis of SLC25A33 and SLC25A36 depletion in the indicated knockout (KO) and double knockout (DKO) HeLa cells (left) alongside the relative mtDNA levels of two monoclonal SLC25A33/SLC25A36 DKO cell lines calculated by qPCR (CYTB / ACTB ;right) (n=3 independent cultures).(D) Representative images taken from the arrayed CRISPR-SpCas9 screen of SLC25A33/SLC25A36 DKO cells showing one field of view from wells transfected with non-targeting control (NTC) sgRNA or TFAM sgRNA (scale bar = 20 µm)(G) Relative mtDNA levels in NME4 KO and NME4/SLC25A33/SLC25A36 triple KO HeLa (left) or NME6 KO and two clones of NME6/SLC25A33/SLC25A36 triple KO HeLa (right). MtDNA calculated by qPCR (CYTB / ACTB) and presented as log2 fold change compared to levels in WT HeLa cells (n = 4-5 independent cultures).(H) Representative immunoblot analysis of the indicated HeLa cell lines (UQCRC2, Ubiquinol-cytochrome C reductase core protein 2; MT-CO2, mitochondrial encoded cytochrome c oxidase II; ATP5A, ATP synthase F1 subunit alpha).
Figure 2(A) The growth rate of NME6 KO and NME6 KO + NME6-MycFlag HeLa relative to WT HeLa cells incubated in DMEM containing 25 mM glucose (log2; n=13 independent cultures).(B) Violin plot of gene effects of NME3, NME4 or NME6 depletion in 1086 cell lines from the Cancer Cell Line Encyclopaedia (CCLE) determined by CRISPR screening (DepMap 22Q2 Public+Score, Chronos; solid line denotes median, dotted line denotes 25 % quartile).(D) Representative live-cell images of the indicated cell lines grown in Human Plasma Like Medium (HPLM) (left) and calculated cell death after 96 h (right). Cell confluency is depicted with the yellow mask and dead cells are identified by SYTOX green staining in purple (n=3 independent cultures).
Figure 3(A) Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) of the indicated HeLa cell lines during a mitochondrial stress test with inhibitor treatments at the indicated timepoints. P-values for OCR: two-way ANOVA P-value (time) = <0.0001, P-value (genotype) = <0.0001, P-value (interaction) = <0.0001; P-values for ECAR:two-way ANOVA P-value (time) = <0.0001, P-value (genotype) = <0.001, P-value (interaction) = 0.0517; P-values for genotype are shown (Oligo, oligomycin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Rot+AA, rotenone and antimycin a; n=3 independent experiments).(B) Heat map representation of z-scores of log2-transformed protein intensities determined by quantitative mass spectrometry and filtered for mitochondrial proteins according to MitoCarta 3.0 The proteins shown clustered together due to their significant (permutation-based FDR < 0.05, s0 = 0.1) depletion in NME6 KO HeLa cells compared to WT and NME6 KO + NME6-MycFlag HeLa cells. OXPHOS subunits are indicated in the right column (n=4 independent cultures).(E) Immunoblot analysis of WT HeLa cells, NME6 KO cells and NME6 KO cells expressing NME6-MycFlag (WT) or NME6H137N-MycFlag (H137N).(F) Immunoblot analysis of WT cells and two NME6 KO clones (#1, #2) generated in three different liver cancer cell lines: HLE, Huh6 and HepG2.(H) The relative growth of WT and NME6 KO liver cancer cell lines monitored on each day (d) using an ATP luminescence assay. P-values for HLE cells: two-way ANOVA P-value (time) = <0.0001, P-value (genotype) = <0.0001, P-value (interaction) = <0.0001; P-values for Huh6 cells: two-way ANOVA P-value (time) = <0.0001, P-value (genotype) = 0.0005, P-value (interaction) = <0.0001;p-values for genotype are shown (n=4 independent cultures).
Figure 4(A) Representative mitochondrial translation assay monitored by the incorporation of 35S methionine and cysteine into the indicated mtDNA-encoded proteins followed by autoradiography (top panel). MtDNA encoded proteins are labelled according to expected size and NME6 and SDHA immunoblots are shown below. (B) Quantification of 35S methionine and cysteine incorporation into all mtDNA-encoded proteins labelled in (B) relative to WT HeLa cells at 15 min (log2; mitochondrial preparations from n=3 independent cultures).(C) Immunofluorescence of NME6 with mtDNA (top) or bromouridine (BrU) labelled nascent mtRNA (bottom) in HeLa cells imaged by confocal microscopy. Relative fluorescence intensities were calculated from linescans generated within the mitochondrial regions indicated by dotted lines.(E) MtDNA-encoded transcript levels analysed by qRT-PCR in NME6 KO and NME6 KO + NME6-MycFlag cells relative to WT HeLa cells. P-values for mtDNA-encoded transcripts: two-way ANOVA P-value (transcript) = <0.0001, P-value (genotype) = <0.0001, P-value (interaction) = <0.0001 (log2; n=3 independent cultures).(F) Nuclear DNA-encoded transcript levels analysed by qRT-PCR in NME6 KO and NME6 KO + NME6-MycFlag HeLa cells relative to WT (log2; n=3 independent cultures).
Figure 5(A) Heat map of log2 transformed mean mitochondrial transcript levels of WT and NME6 KO HeLa cells incubated with 100 µM rNTPs or dNTPs for 48 h relative to untreated WT cells analysed by qRT-PCR (n=3 independent cultures).(B) Volcano plot representation of log2 fold change in proteins between NME6 KO cells treated with 100 µM rNTPs for 120 h and untreated NME6 KO HeLa cells determined by quantitative mass spectrometry. OXPHOS subunits are highlighted in teal (n=3 independent cultures)(D) Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) of WT and NME6 KO HeLa cells incubated with or without nucleosides (100 µM) for a minimum of 120 h. Mitochondrial stress test was performed as in Fig 3A (n=3 independent experiments). P-values for OCR: two-way ANOVA P-value (time) = <0.0001, P-value (genotype) = 0.0011, P-value (interaction) = <0.0001; P-values for ECAR: two-way ANOVA P-value (time) = <0.0001, P-value (genotype) = 0.8994, P-value (interaction) = <0.0001(G) CYTB (top) and ND5 (bottom) transcript levels analysed by qRT-PCR in WT and NME6 KO HeLa cells incubated with the indicated nucleoside species for 48 h. P-values were calculated using a one-way ANOVA (A, adenosine; G, guanosine; C, cytidine; U, uridine; log2; 100 µM; n=4 independent cultures).(H) CTP (top) and dCTP (bottom) levels in the mitochondria enriched fraction of WT and NME6 KO HeLa cells incubated with the indicated nucleoside species for 120 h as determined by quantitative mass spectrometry. P-values for CTP: two-way ANOVA P-value (supplementation) = <0.0001, P-value (genotype) = <0.0001, P-value (interaction) = 0.3179; P-values for dCTP: two-way ANOVA P-value (supplementation) = <0.0001, P-value (genotype) = <0.0001, P-value (interaction) = 0.0569; P-values for supplementation are shown (C, cytidine; U, uridine; log2; 100 µM; n=4 independent cultures).(I) Basal (top) and maximal (bottom) oxygen consumption rates of WT and NME6 KO HeLa cells incubated with the indicated nucleoside species for a minimum of 120 h relative to untreated WT cells (A, adenosine; G, guanosine; C, cytidine; U, uridine; log2; 100 µM; n=4 independent experiments). P-values were calculated using a one-way ANOVA.
Figure 1(E) Representative bands from SDS-PAGE blots of ATPIF1, SDHB, ATP5A1, and Vinculin in control and mutant patient derived fibroblasts. Right panel shows mean of ATPIF1 relative intensity normalized per ATP5A1 and represented as % of control cells (n=2). The colored histogram illustrates the mitochondrial element impacted by each mutation. Note that ATPIF1 expression levels in patient cells is lower than in cells from control subjects. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Unpaired t-test versus Ctrl or untreated conditions shows statistical differences depicted by p-value.(J) State 3 and maximal respiration driven by pyruvate + malate in isolated mitochondria from mouse heart. EPI was added in the respiration media at the indicated concentrations (n≥3). Note that EPI does not affect maximal respiration but has a small effect on ATP synthesis-dependent OCR. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Unpaired t-test versus Ctrl or untreated conditions shows statistical differences depicted by p-value.
Figure 2(C) Representative blot (left) and quantification (right) showing CV assembly by BNGE in mouse heart mitochondria treated with EPI as in A. For each biological replicate, technical replicates were averaged. Data represent average ± SEM.(F) CV in gel ATP hydrolysis in mouse heart mitochondria where EPI or oligo were added in the assay buffer after mitochondrial complexes and supercomplexes were separated by BNGE. CV in gel ATP hydrolysis activity is shown after 3hrs incubation (top). Coomassie staining was used as loading control (bottom). For each biological replicate, technical replicates were averaged.
Figure 3Binding of purified recombinant ATPIF1-GFP to CV. Mitochondrial membranes were disrupted by freeze-thaw and exposed to ATPIF1-GFP at different ratios of mitochondrial (mt) protein/ATPIF1 purified protein. Binding was assessed in BNGE by quantifying ATPIF1-GFP fluorescence. (A) BNGE assessed for ATPIF1-GFP binding (top); CV hydrolytic activity (middle) and protein loading (bottom).Binding of purified recombinant ATPIF1-GFP to CV. Mitochondrial membranes were disrupted by freeze-thaw and exposed to ATPIF1-GFP at different ratios of mitochondrial (mt) protein/ATPIF1 purified protein. (C-D) Representative acidification rate profile showing the effects on ATP hydrolytic activity (C) and its quantification (D) (n=4). For each biological replicate, technical replicates were averaged. Data represent average ± SEM.Binding of purified recombinant ATPIF1-GFP to CV. Mitochondrial membranes were disrupted by freeze-thaw and exposed to ATPIF1-GFP at different ratios of mitochondrial (mt) protein/ATPIF1 purified protein. Binding was assessed in BNGE by quantifying ATPIF1-GFP fluorescence. Binding competition assay between ATPIF1-GFP and EPI to CV. (E) BNGE assessed for ATPIF1-GFP binding (top); CV hydrolytic activity (middle) and protein loading (bottom).