EMBO/SourceData huggingface dataset:
Figure 1A Phase contrast images of Mabs cultured into PF hydrogels (5 days): cells formed a thick three-dimensional myotube network with numerous thick myofibres (arrows). Scale bar: 50 μm.B Confocalimmunofluorescence analysis with an antibody against myosin heavy chain (MyHC) (red) and SYTOX green which labels nuclei (green), showing multi-nucleated, mature musclefibres (arrows) developed from Mabs in PF (5 days). Scale bar: 50 μm.C, D Western blot and densitometry analysis of different protein extracts of Mabs differentiated inside PF constructs (PF) showing expression levels comparable to adult TA (Ctrl). Group of n = 6 of Mabs differentiated inside PF and host TA has been tested, densitometric analysis from n = 3 different Western blots gauging the level of expression of muscle markers is presented as means ± standard error.
Figure 2A Gross morphology of the PF implant containing Mabs-nLacZ/PlGF immediately after implantation on the surface of underlying TA (arrow).B, C Artificial muscles (arrows) developed within 8 weeks over the host TA surface (arrowheads), encapsulated by the host perimysium (B) and released after tendon resection of the TA (C).D The isolated artificial muscle from the TA surface (arrow) is comparable in size to the underlying host TA (arrowhead).E Stereomicroscopy images revealing blood vessel formation in the artificial muscle. Scale bar: 100 μm.F X-Gal and H&amp;amp;E staining on artificial muscle section revealing vessel connection with host vasculature by Indian ink, injected into the femoral artery immediately before sacrifice. Scale bar: 20 μm.G Western blot analysis of protein extracts from artificial muscles (PF) and host TA (Ctrl) reveals remarkable expression of muscle- and vessel-specific proteins (n = 5).I-L X-Gal and H&amp;amp;E staining of artificial muscle sections at 4 (I, K) and 8 weeks (J, L) after implantation, showing donor origin of muscle cells (nLacZ+ nuclei) and correlation between implant duration and myofibre maturation (I, J images are the results of the collage of several photos). Scale bar: (I, J) 200 μm, (K, L) 100 μm.M-P Immunofluorescence analysis on adjacent sections revealing laminin organization (green) and myosin heavy chain (MyHC) expression (red) at 4 weeks (M) and 8 weeks (N) after grafting (M, N displayed items are obtained from a multi-photo collage); DAPI staining (blue) shows centrally located nuclei in developing fibres (O) and peripheral nuclei in mature myofibres (P). Scale bar: (M, N) 200 μm, (O, P) 50 μm.Q Confocal isosurface image of neurofilament (green) and bungarotoxin (red) immunofluorescence in the artificial muscle section shows a mature neuromuscular plaque. Scale bar: 10 μm.R Immunofluorescence against smooth muscle actin (blue), laminin (green) and VE-Cad (red) superimposed on phase contrast image shows blood vessels (arrows) adjacent to the myofibres in the artificial muscle section. Scale bar: 20 μm.S X-Gal staining on mature artificial muscle (arrowhead) and the underneath host TA (arrow) revealing no tendon development at the extremity of the artificial tissue; implanted LacZ-positive Mabs are present within the host TA. Scale bar: 200 μm.
Figure 3A Electron microscopy with Bluo-Gal staining reveals a LacZ-positive donor nucleus (arrow) peripheral to transversely sectioned sarcomeres of the artificial muscle. Scale bar: 1 μm.B Longitudinal EM section of an artificial muscle showing mature sarcomere myofibril organization. Scale bar: 0.5 μm.C A single fibre from an artificial muscle showing a striation pattern typical of a mature myofibre. Scale bar: 10 μm.D X-Gal staining of freshly dissected artificial muscle bundle at 8 weeks showing abundant LacZ-positive donor nuclei. Scale bar: 100 μm.E Immunofluorescence analysis of LacZ-positive nuclei (green) and dystrophin (red) in an individual muscle fibre after functional analysis. Scale bar: 10 μm.F The same fibre shown in (E) is counterstained with DAPI to show that the interstitial nuclei (blue) are of donor origin. Scale bar: 10 μm.G-I Specific force (Po/CSA) (G), maximum shortening velocity (Vo) (H), and cross-sectional area (CSA) (I) of single fibres (n = 153) from artificial muscles and control (CTRL) muscles (n = 103).J Silver-stained SDS-PAGE (8%) gel revealing myosin heavy chain (MyHC)-2B isoform expression in two single fibres of artificial muscles (lanes 1, 2) and a bulk muscle control sample from soleus muscle containing all four adult MyHC isoforms indicated in (K) (lane 3).K Comassie blue-stained SDS-PAGE (8%) of bulk samples of two artificial muscles (lanes 1, 2) and of a bulk control muscle sample (lane 3), as in (J), showing different MyHC isoform expression.
Figure 1B WT and SirT7-/- mice at 10 days (top) and 2 months (bottom) of age.C Weight distribution of WT and SirT7-/- mice (n=3-10 female mice per time point and genotype; P value = 0.003 by unpaired t-Test).Kaplan Meier survival curves (n=170 WT and n=58 SirT7-/- mice; Log-rank Test P value <0.0001).
Figure 2A Representative 3D reconstructed CT scans showing increased kyphosis in 16 month old SirT7-/- mice compared with WT.B Representative gonadal fat pads from WT and SirT7-/- 16 month old mice.C Quantitation of body weight (left) and gonadal fat pad mass normalized to total body weight (right) (mean ± SEM; 4 samples per genotype).D IGF-1 protein levels in serum measured by ELISA (mean ± SEM; 3-8 mice per genotype and age group).E, F Dot plots of Lin-Sca1+cKit+ (LSK) cells (middle and right) from WT and SirT7-/- bone marrow cells. Cells were gated for negative staining of lineage markers B220, CD3, CD11b, CD19, Gr-1, and Ter-119 (Left), and analyzed for Sca1 and cKit expression (middle and right). (F) Quantitation of (E) (mean ± SEM; 4 mice per genotype).G Bone marrow, thymus, and spleen cell number in young WT and SirT7-/- mice (mean ± SEM; 3-5 mice per genotype).H mRNA expression of p16 gene normalized to GAPDH measured by RT-PCR from young and old WT and SirT7-/- fibroblasts (mean ± SEM; 3 samples per genotype).I, J Senescence-associated β-galactosidasestaining (I, blue) in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and grown for 7 days in selection media. (J) Quantitation of the number of senescent cells shown in (I) (mean ± SEM; 3 independent cell lines per genotype).*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by ANOVA Single Factor.
Figure 3A, B Dot plots of FACS cell cycle analyses of WT and SirT7-/- MEFs in passage 3 (P3) and 6 (P6) using EdU incorporation and 7AAD (A). Percentages of cells in Sub G1 (apoptotic cells, left square) and cells with DNA content above 4N (polyploid, right square). (B) Quantitation of experiment shown in (A) (mean ± SEM; 3 samples per genotype).C Survival curve for WT and SirT7-/- thymocytes after X-Ray irradiation (IR) at the indicated doses. Cell death was quantified by FACS using AnexinV and 7AAD staining 18 hours post insult (mean ± SEM; 3 samples per genotype from one of two independent experiments).D Clonogenic assays in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and irradiated at the indicated X-Ray doses, then plated at low density. After 9 days, colonies were stained with crystal violet and counted (mean ± SEM; 3 independent cell lines per genotype).F Quantitation of experiment shown in (E) (mean ± SEM; 9 WT and 3 SirT7-/- samples).*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.
Figure 4A Quantitation of neutral comet assays, using passage 3 WT and SirT7-/- primary MEFs, showing (left) the amount (%) of DNA in the tail and (right) the tail moment (see Fig S3A for representative images).B Western blot showing ATM and KAP1 phosphorylation and total protein levels in WT and SirT7-/- primary fibroblasts after IR (8Gy). ATM inhibitor (ATMi) KU-55933 was added 30 min prior to irradiation where indicated. One representative blot from 4 independent experiments is shown.C-F IF analysis of WT and SirT7-/- primary fibroblasts showing γH2AX dynamics after DNA damage induction. Cells were untreated (NoIR) or treated with 1Gy of X-rays (IR) and fixed at the indicated times post insult. Cells were pulsed with EdU (green) 30 min prior to fixation, then stained for γH2AX (red) and counterstained with DAPI (blue) (n>30 cells per group/mice; 3 mice per genotype). (C) Representative images of untreated S-phase (NoIR, EdU positive) WT and SirT7-/- nuclei (scale bar 5µm). (D) Quantitation of the number of γH2AX foci per nucleus at the indicated time points post IR (1Gy), and indicated cell cycle phases. (E) Quantitation of experiment described in (C) showing mean number of γH2AX foci per nucleus in euchromatic and heterochromatic regions at the indicated time points before and after IR in G2 (mean ± SEM from 3 independent experiments). Total γH2AX foci and γH2AX foci overlapping with or at the periphery of heterochromatic regions were enumerated. Nuclei and pericentric heterochromatin (chromocenters) were segmented by DAPI staining. Euchromatic numbers were estimated by subtracting the heterochromatic number of foci from the total foci number.(F) Representative images of WT and SirT7-/- primary fibroblasts in G2 phase showing γH2AX (red) and DAPI (blue) at the indicated period of time after IR. (Right) 3D rendering of the IF segmentation depicting nuclei (pale blue), chromocenters (darker blue), and γH2AX (yellow denotes foci associated with pericentric heterochromatin, otherwise foci are red). Scale bar 2 µm.G FACS quantitation of WT and SirT7-/- MEF cells in S-phase after insult with 10 mM hydroxyurea (HU) for 24 hours. Cells were fixed and stained with 7AAD and cell cycle was monitored by FACS (mean ± SEM; 5 samples per genotype).H, I DNA fiber labeling analysis was used to assess DNA replication fork progression in passage 3 and passage 6 primary WT and SirT7-/- MEFs. (H) Representative images from cells labeled for 20 min with IdU (green) followed by 20 min of CldU (red). (I) Quantitation of fork velocity (fiber length/labeling time; mean ± SEM; 3 samples per genotype per condition).*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.
Figure 5A-E IF analysis of WT and SirT7-/- primary fibroblasts after IR (1Gy) and 1 hour chase. Cells were pulsed with EdU 30 min prior to fixation, stained with antibodies against γH2AX and 53BP1 and then counterstained with DAPI (scale bar 5µm). (A) Representative images from each cell cycle stage showing merge (top-left) including EdU (green), 53BP1 (top-right, magenta), γH2AX (bottom-left, red), and 3D rendering of reconstructed Z-stacks with 53BP1 foci modeled as spheres (bottom-right; pale-blue: nucleus; magenta spheres: 53BP1 foci). (B) Quantitation of the number of 53BP1 foci per nucleus, and (C) mean volume of 53BP1 foci (n>30 cells per group/mice; 3 mice per genotype). (D-E) Same as in (B-C), upon overexpression of SIRT7 WT or catalytically inactive point mutant SIRT7-H188Y in WT and SirT7-/- primary fibroblasts.F Detection of endogenous chromatin-bound and nucleoplasmic 53BP1 protein from WT and SirT7-/- MEFs before and after IR (8Gy) by western blot. Histone H3 was used as loading control, and tubulin as control for fractionation.G Same as (F), using 293T-REX cells treated with doxycycline or vehicle to induce SIRT7-HA expression.H ChIP-on break assay. (Top) Schematic of I-SceI substrate construct introduced into HT1080 cells, which contains a single I-SceI site located within a puromycin resistance cassette. Shown along the top are the coordinates for amplicons probed by Q-PCR. (Bottom) 53BP1 enrichment at the indicated loci in Scramble control or SirT7 knockdown cells. Q-PCR measurements were normalized to input DNA and non-I-SceI treated samples (No Cut) (mean ± SEM; one of two independent experiments shown). (Bottom Right) western blot demonstrating efficient knockdown of SirT7, with Histone H3 as a loading control.I, J NHEJ repair assay using GFP expression-based reporter system in SirT7-depleted HT1080 cells (SirT7 Knockdown) versus control (Scramble Control). (I) Representative FACS dot plots. (J) Quantitation of (I); mean ± SEM of 3 independent clones per condition. Values are normalized to control (Scramble Control + I-SceI) mean.K, L Class-switch recombination in splenic B cells from WT and SirT7-/- mice stimulated with lipopolysaccharides (LPS) and interleukin 4 (IL4). (K) The switching from IgM to IgG1 (left) and to IgG3 (right) was measured by FACS. (L) Quantitation of (K) showing mean ± SEM of 3-4 samples per genotype. One representative experiment from two is shown.*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.
Figure 6A, B H3K18Ac IF in WT and SirT7-/- fibroblasts. Cells were pulsed with EdU (green), stained with antibody against H3K18Ac (magenta) and then counterstained with DAPI (blue). (A) Representative IF images of late S-phase nuclei (EdU positive; scale bar 5µm). (B) Quantitation of (A) throughout the cell cycle.C Same as in (B), in S-phase cells at the indicated times after IR (1Gy) (mean ± SEM of >30 cells per cell cycle, time point, and genotype).D Laser-induced DNA damage in HT1080 cells expressing HA-tagged SirT7 stained with antibodies against γH2AX (red) and HA (green), then counterstained with DAPI (blue) 30 min post damage (scale bar 3 µm).E ChIP assays of SIRT7 enrichment at the indicated loci as described in Figure 5H (mean ± SEM; one of two independent experiments is shown).F Recruitment kinetics of GFP-tagged SirT7 after laser-induced microirradiation. (Top) Representative cell at the indicated times after induction of DNA damage (white circle; scale bar 2µm). Images were adjusted to account for photobleaching by normalizing to nucleoplasmic background signal. (Middle) Quantitation of recruitment kinetics at the site of induced damage in the presence of 5M KU-55933 ATM inhibitor (ATMi), 10M Olaparib PARP inhibitor (PARPi), or DMSO. KU-55933 and Olaparib were added 30 min and 1 hour respectively prior to DNA damage (mean ± SEM; sample size: SIRT7-GFP, n=34; SIRT7-GFP + ATMi, n=36; SIRT7-GFP + PARPi=18). (Bottom) Same as (middle) except quantitation of SIRT7-GFP relative intensity within the nucleolus. Data was acquired at five second intervals over a span of five min.G ChIP assays of H3K18Ac enrichment at the indicated loci as described in Figure 5H. H3K18Ac enrichment adjacent to the induced DSB in control and SIRT7 depleted cells (bottom left), or in cells overexpressing SIRT7 in the presence or absence of PARP inhibitors (PARPi) (bottom right) (mean ± SEM; one of two independent experiments is shown).*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by ANOVA Single Factor.
Figure 7A-H NHEJ activity in HT1080 or NIH3T3 cells stably overexpressing H3-WT, H3-K18Q or H3-K18R (naive, acetylated or deacetylated H3K18 residues, respectively).A Random integration assay in HT1080 cells using a linearized pSMCV vector containing a puromycin resistance cassette (mean number of puromycin resistant colonies normalized by plating efficiency ± SEM; One representative transfection from 3 is shown).B NHEJ repair assay using GFP expression-based reporter system described in Fig 5I (mean ± SEM of 3 independent experiments. See Fig EV5B for representative FACS dot plots).C-D IF staining of NIH3T3 cells after IR (1Gy) and 1 hour chase. Cells were pulsed with EdU 30 min prior to fixation, stained with antibodies against γH2AX and 53BP1, and counterstained with DAPI. Representative images can be found in Fig EV5C. (C) Quantitation of the number of 53BP1 foci per nucleus, and (D) mean volume of 53BP1 foci (mean ± SEM of >30 cells per condition and cell cycle stage).E Western blot analysis of 53BP1 in chromatin and non-chromatin fractions from NIH3T3 cells expressing H3-WT, H3-K18Q or H3-K18R vectors and exposed to IR (10Gy). One representative blot is shown from 3 independent experiments. Shown is 53BP1, GAPDH for fractionation control, and H3 for loading control.F-H Same as in (C-D) except using antibodies against γH2AX (red) and RIF1 (magenta). (F) Representative images from cells in S-phase (EdU positive; scale bar 5µm). (G) Quantitation of the number of RIF1 foci per nucleus, and (H) mean volume of RIF1 foci (mean ± SEM of >30 cells per condition and cell cycle stage).I 53BP1 enrichment at the indicated loci as described in Figure 5H in Scramble control or SirT7 knockdown cells expressing the indicated H3 constructs (mean ± SEM; one of two independent experiments).J Western blot analysis of chromatin fractions from primary WT and SirT7-/- MEFs expressing the indicated H3 constructs and exposed to IR (10Gy). Shown are levels of 53BP1, SIRT7, H3-Myc, GAPDH for fractionation control, and H3 for loading control.
Figure 1Restriction enzyme diagnostic test for the presence of the R749H mutation.qRT-PCR with primers specific to both wild-type and mutant RNAPII (left panel) or to the mutant form of RNAPII (right panel), confirming that only the slow version of RNAPII is expressed in homozygous slow/slow ESCs. The sequences of the respective forward primers are shown. The "WT/slow allele" primer is complementary to the sequence in exon 14 upstream of the mutation. The "slow allele only" primer has its 3' end matching the mutated codon 749 and does not anneal to the WT DNA sequence. The mean ± SEM is plotted, n=2.
Figure 3Meta-gene profile of normalized 4sU-DRB-seq reads in WT/WT and slow/slow ESCs.Density and violin plot of elongation rate (bases/min) calculated for genes common in all genotypes in WT/WT, WT/slow and slow/slow ESCs. Box
Figure 4Brightfield images and analysis of NPC markers by immunofluorescence staining (Sox1 and Nestin) or RT-qPCR (Nestin and Pax6) (n=3, mean ±SEM).Brightfield images of aggregates.Brightfield images and immunofluorescence staining for Nestin and neuronal marker Tuj1 in NSC cultures grown in EGF/FGF proliferating conditions. White arrows indicate NSCs and green arrows differentiated cells. Two small panels on the right are examples of Tuj1+ neuronal cells in slow/slow NSCs cultures.Immunofluorescence staining for neuronal marker Tuj1 in neuronal cultures grown on poly-ornithine/laminin at 21 days of differentiation.
Figure 5RT-PCR analysis validation of selected alternatively spliced exons. RT-PCR was performed on total RNA from WT or slow/slow ESCs, NPCs or neurons. PCR products were visualized and quantified by Bioanalyzer (Agilent). Images are representative of experiments performed in triplicate. Themean ± SEMis plotted with *p-value<0.05, **p<0.005, ***<0.0001 as determined by t-test.
Figure 6Box plot showing the length of upregulated (UP), downregulated (DOWN), not affected genes (ALL), and pre-mRNAs affected by alternative splicing (AE, for Alternative exons) for ESCs (left panel); length of upregulated (UP), downregulated (DOWN), not affected genes (ALL), pre-mRNAs affected by alternative splicing (AE, for Alternative exons), and not affected genes that are only expressed in neurons (but not in ESCs) (ALL_notESC), for Neurons (right panel). n.s., p-value>0.05, **p<0.005; ****p<0.0001 as determined by Mann-Whitney t-test. Boxes delimit the first and third quartiles. The horizontal lines represent the data medians. Whiskers are drawn down to the 1st and 99th percentile. Percentage of genes that are downregulated in ESCs and neurons; plotted as a sliding window of 100 genes by length.
Figure 1(A) Number of cell corpses at the L1 stage of development per 100 animals carrying the neurotoxic mec‐4(d) or deg‐3(d) alleles in SNT‐1‐deficient mutants. For mec‐4(d) mutant animals, bars denote touch receptor neuron corpses. For deg‐3(d)mutants, bars denote inner labial neuron 1 (L1) sensory neuron and PVC interneuron corpses.(B) Expression of a full‐length MEC‐4::GFP reporter fusion under the control of the mec‐4 promoter in touch receptor neurons of wild‐type, snt‐1 and unc‐57 mutant animals. Representative photos of touch receptor neuron cell bodies are shown. Bar denotes 6 μm. The quantification (%) of GFP signal intensity from the animals examined is graphed below (n=100 neurons per assay; P0.05 compared with wild type; t‐test).(C) Percentage of SNT‐1‐deficient animals that survive near‐lethal treatment with sodium azide. This chemical inhibits the activity of the respiratory electron transport complex IV (cytochrome C oxidase) and simulates hypoxia.(D) Number of touch receptor neuron corpses, at the L1 stage of development, per 100 animals carrying the neurotoxic mec-4(d) or deg‐3(d) alleles in UNC‐57‐deficient animals.(E) Percentage of UNC‐57‐deficient animals that survive after the hypoxia‐inducing treatment with sodium azide.(F) Number of touch receptor neuron corpses, at the L1 stage of development, per 100 animals carrying the neurotoxic mec‐4(d) or deg‐3(d) alleles together with lesions in genes encoding key proteins that participate in all four steps of clathrin‐mediated endocytosis. UNC‐11 and DPY‐23 are adaptor proteins required for the formation of clathrin‐coated pits, DYN‐1 is a GTPase necessary for fission of clathrin‐coated vesicles and UNC‐26 participates in the uncoating of vesicles. Error bars denote s.e.m. values (n>250 for all populations examined; P0.001, compared with wild‐type animals, unpaired t‐test).
Figure 2(A) Confocal images of touch receptor neurons expressing a pmec‐17APS‐2GFP reporter transgene. During early necrosis, the number of APS‐2::GFP puncta, corresponding to clathrin‐coated pits and vesicles, in touch receptor neurons ofmec‐4(d) animals does not change significantly, whereas at later stages it decreases. A similar decrease is also observed in unc‐57(e1190) mutants, defective for clathrin‐mediated endocytosis.(B) Confocal images of touch receptor neurons expressing a pmec‐7GFP::RAB‐5 reporter transgene. The number of fluorescent puncta that correspond to early endosomes in touch receptor neurons of mec‐4(d) animals significantly increases during early necrosis, whereas it declines later.(C) Confocal images of touch receptor neurons expressing a pmec‐7GFP::RAB‐11 reporter transgene. The number of GFP::RAB‐11 puncta that correspond to recycling endosomes in touch receptor neurons of mec‐4(d) animals increases early during cell death, while it declines as degeneration proceeds. Both early and recycling endosomes tend to accumulate around a swollen structure that is probably the nucleus. Bars denote 4 μm. Error bars denote s.e.m. values (n>250 for all populations examined; P0.001, compared with control animals, unpaired t‐test).
Figure 3Endocytosis functions together with lysosomal proteolysis to facilitate necrotic cell death. (A) Depletion of synaptotagmin (SNT‐1) or endophilin (UNC‐57) does not further suppress MEC‐4(d)‐induced necrosis in cad‐1 mutants with reduced cathepsin activity or with V‐ATPase dysfunction (B, C).Similarly, no synthetic reduction of necrotic cell death is observed in calpain‐deficient animals that also carry lesions in the synaptotagmin (D) or endophilin (E) genes. Error bars denote s.e.m. values (n>250 for all populations examined; P>0.5, compared with single mutant control animals, unpaired t‐test).
Figure 4(A) Synaptotagmin (SNT‐1) deficiency further suppresses MEC‐4(d)‐induced necrosis in animals with impaired autophagy.(B) Endophilin dysfunction enhances suppression of necrosis in mec‐4(d);lgg‐1(RNAi) animals, where autophagy is impaired. Error bars denote s.e.m. values (n>250 for all populations examined; P0.01, compared with single mutant control animals, unpaired t‐test).