EMBO/SourceData huggingface dataset:
Figure 5Numbers of GFAP+BrdU+ label-retaining NSCs in the V-SVZ. Arrowheads point to label-retaining NSCs. n=4, *p= 0.039. V-SVZ coronal sections stained for GFAP (cyan) and BrdU (red). Scale bar: 10 μm.Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 24h after TMZ treatment. Arrowheads point to dividing NSCs under regenerative condition. n=6, *p= 0.0173. Scale bar: 10 μm.Densities (cells/mm2) of DCX+ neuroblasts in V-SVZ whole-mounts 3 days after TMZ treatment. n=3, *p= 0.0179. V-SVZ whole-mounts stained for DCX. Scale bar: 500 μm.Densities (cells/mm2) of BrdU+ cells in the OB GCL labeled 24h after TMZ treatment. n=5 (Ctrl) and 6 (Ccn1cKO), *p= 0.0484. OB sections stained for BrdU (yellow) and nuclei (blue). Scale bar: 50 μm.Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts of aged mice 24h after TMZ treatment. Arrowheads point to dividing NSCs under regenerative condition. n=3, *p= 0.0321. Scale bar: 10 μm.Densities (cells/mm2) of DCX+ neuroblasts in V-SVZ whole-mounts of aged mice 3 days after TMZ treatment. n=3, n.s. not significant. Scale bar: 500 μm.Densities (cells/mm2) of BrdU+ cells in the OB GCL of aged mice labeled 3 days after TMZ treatment. n=4 (Ctrl) and 3 (Ccn1cKO), n.s. not significant. Scale bar: 50 μm.
Figure 6Representative FACS plots showing gating strategy for the analysis of cell types in the adult V-SVZ.Total number of V-SVZ cells isolated from one mouse. n=3, n.s. not significant.Representative FACS plots showing aNSCs (GFP+EGFR+), qNSCs (GFP+EGFR-) and C cells (GFP-EGFR+) in the adult V-SVZ.Percentage of each cell population in the adult V-SVZ. n=3, **p=0.0014 (GFP+EGFR-), ***p=0.0002 (GFP+EGFR+), *p=0.0111 (GFP-EGFR+), ***p=0.0006 (GFP+), n.s. not significant.Densities (cells/mm2) of VCAM1+ B1 cells in the adult V-SVZ whole-mounts. n=4 (Ctrl + vehicle and Ccn1cKO + vehicle) and 3 (Ccn1cKO + erlotinib), **p=0.0021 (Ccn1cKO + vehicle vs Ccn1cKO + erlotinib), **p=0.0014 (Ccn1cKO + erlotinib vs Ctrl + vehicle), ****p<0.0001.Densities (units/mm2) of NSC units in the adult V-SVZ whole-mounts. n=4 (Ctrl + vehicle and Ccn1cKO + vehicle) and 3 (Ccn1cKO + erlotinib), *p=0.017, **p=0.0061, ***p=0.0002.V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white). Scale bar: 50 μm.
Figure 1 A Neonatal rat ventricular myocytes (NRVMs) were infected with the 3xMEF2-Luc reporter, serum starved for 20 hours and stimulated for 24 hours with different agonists: 100 nM endothelin-1 (ET1), 1 μM sphingosine-1-phosphate (S1P), 10 μM lisophosphatidic acid (LPA), 1 μM WIN55,212-2 (WIN55, cannabinoid receptor agonist), 1 μM isoproterenol (ISO, β-adrenergic receptor agonist), 100 nM angiotensin II (AngII), 10 μM prostaglandin E1 (PGE1), 10 μM prostaglandin E2 (PGE2) or 100 nM prostanoid F receptor agonist fluprostenol (Flupro), 100 nM treprostinil (Trepro, prostacyclin receptor agonist) Data information: values are mean±s.e.m. In (A), the experiment was performed in triplicates, similar results were obtained in 3 different experiments. Student's two-tailed t-test, *,P<0.05 vs. control (ET1, P=0.0013; S1P, P=0.0144; LPA, P=0.026; WIN55, P=0.6205; ISO=0.0399; AngII, P=0.5812; PGE1, P=0.001; PGE2, P<0.0001; Flupro, P=0.0846; Trepro, P=0.1958)  B PGE2 induces the expression of MEF2 target genes. NRVMs were serum-starved for 24 hours, then were stimulated with 1 μM PGE2 for 2 hours and mRNA levels of the indicated genes were determined Data information: values are mean±s.e.m In (B), n=5, technical replicates, Student's two-tailed t-test, *,P<0.05 vs. control (Nur77, P=0.0148; Myomaxin, P=0.0417; Adamts1, P=0.0112; BNP, P=0.0065) C Correlation between MEF2 activation and NRVM hypertrophy. MEF2 activity was measured as in (A) upon increasing concentration of PGE2. Cell size of NRVMs was determined after 48-hour stimulation with the indicated concentrations of PGE2. Left: representative images of α-actinin stained NRVMs stimulated with DMSO, 300 nM PGE2, or 1 nM ET1. Scale bar is 10 µm Data information: values are mean±s.e.m In (C), NRVM cell size was determined from a minimum of 100 cells of 3 technical replicates upon different concentrations of PGE2, MEF2-activity was parallel assessed from 6 technical replicates. Correlation was statistically analyzed by calculating Pearson correlation coefficient (Pearson r=0.9626, P=0.0021) D, PGE2 activates MEF2 via EP3 receptor. NRVMs were infected with the 3xMEF2-Luc reporter and serum-starved for 20 hours. The cells were stimulated with DMSO or 1 μM PGE2 for 24 hours in the presence or absence of different EP receptor antagonists: AH6809 (10 μM, EP1- and EP2-antagonist) or 798106 (200 nM, EP3-antagonist) or L161,982 (2 μM, EP4-antagonist) Data information: values are mean±s.e.m In (D), n=3, independent experiments, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, AH6809, P=0.1092; L798106, P=0.0002; L161,982, P=0.5579). The exact n and P values can be also found in the Source Data excel file for Figure 1
Figure 2 B-D (B, D) NRVMs were infected with the 3xMEF2-Luciferase reporter and with recombinant adenoviruses encoding EGFP (AdGFP), RGS16, RGS2, p63ΔN, RGS-LSCII, or Gαt, as indicated. The cells were serum starved for 20 hours and stimulated with DMSO or 1 μM PGE2 for 24 hours. (C) NRVMs were infected with the 3xMEF2-Luciferase reporter and pretreated with the adenylyl cyclase inhibitor SQ22536 (100 μM) for 20 minutes or the Gi/o-protein inhibitor pertussis toxin (PTX, 100 ng/ml) for 20 hours in serum-starved conditions and treated with DMSO or 1 μM PGE2 for 24 hours. Data information: In (B-D), n=3, independent experiments, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, RGS16, P<0.0001; RGS2, P=0.1725; p63ΔN, P=0.0666; RGS-LSCII, P=0.0818, SQ22536, P=0,385; PTX, P<0.0001; Gαt, P<0,0001), values are mean±s.e.m. The exact n and P values can be also found in the Source Data excel file for Figure 2
Figure 3 A,B NRVMs were infected with recombinant adenovirus encoding Flag-HDAC5 (A) or not infected (B) and serum starved for 20 hours. The cells were pretreated with the PKC-inhibitor bisindolylmaleimide I (2 μM) or the PKD-inhibitor BPKDi (3 μM) for 20 minutes and stimulated with 100 nM endothelin-1 (ET1) or 100 μM phenylephrine (PE) or 1 μM PGE2 for 4 hours, as indicated. HDAC phosphorylation was detected by immunoblotting with the anti-HDAC5 phospho-Ser-498 antibody, phosphorylation of PKD was assessed using anti-phospho-PKD antibodies (Ser-744/Ser-748 and Ser-916), representative images are shown from 3 independent experiments  C NRVMs were infected with the 3xMEF2-Luc reporter and serum-starved for 20 hours. The cells were pretreated with the PKD-inhibitor BPKDi (3 μM) or the CamKII-inhibitor AIP (1 μM) for 20 minutes and stimulated with DMSO or 1 μM PGE2 for 24 hours. N=3, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, BPKDi, P<0.0001; AIP, P=0.2197), values are mean±s.e.m. The exact n and P values can be also found in the Source Data excel file for Figure 3
Figure 4 A NRVMs were serum-starved for 24 hours and stimulated for 2 minutes with 10 μM PGE2. Rac1 and RhoA GTPase activation was determined by effector pulldown assay and analyzed by immunoblot Data information: In (A-C) representative images from at least 3 independent experiments are shown  B NRVMs were infected with recombinant adenoviruses encoding EGFP (AdGFP) or RGS16 24 hours prior to stimulation, then Rac1 activity was measure Data information: In (A-C) representative images from at least 3 independent experiments are shown. In (B-D) values are mean±s.e.m., (B,C) n=4 3; *,P<0.05 (Two-Way ANOVA, RGS16, P=0.0415 (B ), NSC effect on Rac1 activation, P=0.0412 (C); NSC effect on MEF2 induction, P<0.0001. PGE2 effect was significant in Rac1 activation, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 4 C Cells were pretreated with 100 ng/ml pertussis toxin (PTX, Gi/o-inhibitor) for 24 hours or with 50 μM NSC23766 (NSC, Tiam1-Rac1 interaction inhibitor) for 2 hours. Thereafter Rac1 activation was determined by effector pulldown assay Data information: In (A-C) representative images from at least 3 independent experiments are shown In (B-D) values are mean±s.e.m., (B,C) n=4 3; *,P<0.05 (Two-Way ANOVA, RGS1 PTX, P=0.039 (C), NSC effect on Rac1 activation, P=0.0412 (C); NSC effect on MEF2 induction, P<0.0001. PGE2 effect was significant in Rac1 activation, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 4 D NRVMs were transduced with the 3xMEF2-Luc reporter and were serum-starved for 20 hours. After 2-hour of 50 μM NSC23766 (A) pretreatments, the cells were stimulated with DMSO or 1 μM PGE2 for 24 hours, then MEF2 activity was determined. NSC abolished the MEF2 activation by PGE2 Data information In (B-D) values are mean±s.e.m. (D), n=3; *,P<0.05 (Two-Way ANOVA, RGS1
Figure 5 A After serum starvation NRVMs were pretreated with or without 50 μM NSC23766 for 2 hours and stimulated with 1 μM PGE2 for 1 hour. Total and phosphorylated PKD levels were analyzed by immunoblot. PKD was activated by PGE2, but it was not affected by NSC Data information: In (A-C) representative images from 3 independent experiments are shown  B After infection with Flag-HDAC5 and serum starvation, NRVMs were treated with 50 μM NSC23766 for 2 hours prior to stimulation. The cells were stimulated with 1 μM PGE2 for 1 hour. HDAC5 phosphorylation at Ser-498 was detected by immunoblot. The phosphorylation of HDAC5 at this site was not prevented by NSC Data information: In (A-C) representative images from 3 independent experiments are shown  C NRVMs and neonatal cardiac fibroblasts (CFs) were serum-starved, pretreated with vehicle or 200 nM L798106 (EP3 receptor antagonist) for 20 minutes, and were stimulated with 1 μM PGE2 for 10 minutes. PAK2 and PKD phosphorylation was assessed by immunoblot. Calsequestrin was used as a myocyte-marker Data information: In (A-C) representative images from 3 independent experiments are shown  D MEF2 activity was determined in cells infected with the 3xMEF2-Luc reporter after serum starvation. Cells were stimulated with vehicle or 1 μM PGE2 for 24 hours after 1-hour of 30 μM IPA-3 pretreatment. IPA-3 inhibited the MEF2 activation by PGE2 Data information In (D) values are mean±s.e.m., n=3, biological replicates, *,P<0.05 (two-way ANOVA, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 5
Figure 6 Top: GFP-HDAC5 infected NRVMs were serum-starved for a day, pretreated for two hours with the indicated compounds (BPKDi, 3 µM, IPA-3, 30 µM) alone or in combination, then were stimulated for 4 hours. Representative images. Scale bar is 10 µm. Bottom : Statistical analysis of cellular localization of HDAC5. GFP-HDAC5 localization (nuclear or cytosolic) was assessed in >100 cells of each sample (n=3, technical replicates). *, P<0,05 (one-way ANOVA with Bonferroni post-hoc test, vehicle vs. ET1 or vs. PGE2, P<0.0001; PGE2 vs. PGE2+BPKDi, P=0.0004; PGE2 vs. PGE2+BPKDi, P=0.0114; PGE2 vs. PGE2+BPKDi+IPA-3, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 6
Figure 2A Isothermal calorimetry (ITC) assays using ADP-ribose ligand and purified macrodomains of human macroH2A1.1 and macroH2A2.B In vitro PARP1 activity assay in the presence of increasing concentrations (10, 25 and 50 μM) of purified macrodomains of human macroH2A1.1, macroH2A1.1 G224E mutant and macroH2A2. PARP1 activity is assessed by its auto-PARylation detected by an anti-PAR immunoblot. The naphtol blue staining shows the increasing amounts of purified macrodomain added to each reaction. A representative blot of one of three independent experiments is shown.
Figure 3A The diameter (d) of the fluorescence signaling of H2B tagged with photo-activatable GFP (PAGFP-H2B) is used to quantify chromatin expansion across 120 seconds after induction of DNA damage using intercalating DNA dye and local irradiation with a laser.B Chromatin expansion in absence and presence of intercalating DNA dye (- and + DNA damage) and pretreatment of 1h with 1μM of Olaparib PARP inhibitor. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage from 3 biological replicates with n > 30 cells each. The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values (*, p < 0.05 using unpaired, two-tailed Student's T-test).C Quantified chromatin expansion in cells not transfected (control) and cells transfected with each mCherry-tagged histone (as indicated). Data is represented as in B (n > 30 cells, 3 bio. repl.; *, p < 0.05 compared to mCherry-H2A-expressing cells using unpaired, two-tailed Student's T-test).D Chromatin expansion in Hela cells containing a doxocyclin (doxo.)-inducible macroH2A1.1 transgene. Chromatin expansion assay was performed 48 hours after induction and expression was controlled by anti-macroH2A1.1-immunoblotting. Data is represented as in B (n > 30 cells, 3 bio. repl.; *, p < 0.05 using unpaired, two-tailed Student's T-test).
Figure 4Quantified chromatin expansion in cells transfected with indicated histone constructs. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage (n > 30 cells, 3 bio. repl.). The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test comparing to H2A; n.s., not significant).Quantified chromatin expansion in cells transfected with indicated histone constructs. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage (n > 30 cells, 3 bio. repl.). The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test comparing to H2A; n.s., not significant).
Figure 5A Western blot showing the expression of macroH2A proteins in control HepG2 cells, knock-down for macroH2A1 and macroH2A2 (DKD) and DKD with ectopic expression of the indicated YFP-tagged constructs. H3 is used as a loading control.B H3K9me3 immunostaining in the indicated HepG2 cell lines with nuclear counterstaining in DAPI. The indicated areas are zoomed-in for a better observation of the H3K9me3 signal profile. All images are maximum intensity Z-projections of confocal stacks.Quantification of the nuclear area The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test; n.s., not significant).Quantification of H3K9me3 foci per nucleus, The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test; n.s., not significant).
figf1Wild-type, apg12-1 mutant and Δapg12 cells were cultured in nitrogen-starvation medium containing 1 mM PMSF. After incubation for 6 h, cells were observed under a phase-contrast microscope. Arrows indicate autophagic bodies.c, Wild-type (squares) and Δapg12 (circles) were cultured in nitrogen-starvation medium and their viability was determined by phloxine B staining.d, Quantification of autophagic activity of wild-type and Δapg12 cells by alkaline phosphatase (ALP) assay before (black bars) and after (white bars) nitrogen starvation for 4 h. Error bars indicate s.d. of three independent experiments.
figf2a, b, Lysates from Δapg12 cells carrying only vector or 3 × HA-APG12 (a), and Δapg5, apg7-1, apg10-1 and Δapg1 cells carrying 3 × HA-Apg12 plasmid (b) were immunoblotted using anti-HA antibody. The positions of 3 × HA-Apg12 and the larger product (asterisks) are indicated.c, Immunoblot analysis of wild-type and Δapg12 cells harbouring HA-APG5 plasmid.d, Δapg5 Δapg12 cells were co-transformed with Myc-APG12 and HA-APG5. Their lysates were immunoprecipitated with anti-Myc or anti-HA antibodies and detected by immunoblotting using anti-Myc antibody. The position of the crossreacting IgG heavy chain is indicated
figf3b, Δapg12 cells were transformed with the mutant plasmids and their lysates were immunoblotted with anti-HA antibody.c, Autophagic activity was measured as described for Fig. 1d.d, Transport of pro-API to the vacuole was examined by immunoblotting with anti-API antiserum. The positions of pro-API and mature API are indicated.
figf4Δapg5 cells were transformed with vector alone, wild-type APG5 or APG5K149R, and then immunoblotted with anti-HA (b)Autophagic activity was determined by alkaline phosphatase assay (c).Δapg5 cells were transformed with vector alone, wild-type APG5 or APG5K149R, and then immunoblotted anti-API (d).
figf5c, In vitro conjugation of Apg5 and Apg12. A cell lysate of Δapg12 carrying HA-APG5 (lane 1) was incubated with an equal amount of lysate from Δapg5 carrying HA-APG12 (2 µ plasmid) (lane 2) at 30 °C with (lane 4-6) or without (lane 7) 5 mM ATP. Samples were mixed with SDS-sample buffer at the times indicated.
figf6Spheroplasts were generated from cells expressing either HA-Apg12 or HA-Apg5. Their lysates were mixed and layered on top of a 10-step (18-54 % w/w) sucrose gradient, and centrifuged at 174,000g for 2.5 h (ref. 29). Fifteen fractions were collected and the positions of free Apg5, free Apg12 and Apg5/Apg12 conjugate were examined by western blotting. The peak fractions of alcohol dehydrogenase (ADH)(cytosol), ALP (vacuole), Kex2 (Golgi) and Sec12 (endoplasmic reticulum) are indicated by arrows.
Figure 1A) We identified previously unknown telomere regulators in fission yeast using the haploid S. pombe whole-genome gene deletion library including ssu72+ (SPAC3G9.04). B) Telomere length of wt, ssu72Δ and ssu72-C13S (point mutation at the phosphatase active site) strains were measured by Southern blot in ApaI digested genomic DNA using a telomeric probe. C) Ssu72 recruitment to telomeres is cell cycle regulated. Ssu72 was myc-tagged at the 3' end and ChIP analysis was carried out in cell cycle synchronized populations of cdc25ts strains. Septa formation was used proxy for S-phase. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to ssu72+ control samples. Error bars represent standard error of the mean (SEM).D) Telomere length of ssu72Δ is dependent on telomerase. Diploid strains with the appropriate genotypes were sporulated and trt1∆ ssu72∆ double mutants were streaked for multiple passages (triangle indicates increased number of generations).E) Telomerase is recruited to telomeres in the absence of Ssu72. ChIP analysis for Trt1-myc in wt and ssu72∆ was performed as described in material and methods using a non-tagged strain as a control. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).F) Telomerase activator Ccq1 is hyperphosphorylated in ssu72∆ cells. rap1∆ cells were used as positive control for Ccq1 phosphorylation status. Western blots were performed using Ccq1-flag tagged strains.
Figure 2A) ssu72Δ telomeres present longer G-rich overhangs than wt and rif1∆. In-gel hybridization in native and denaturing conditions was labelled with a radiolabelled C-rich telomere probe and quantified for ssDNA at the telomeres. n = 3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).B) RPA (Rad11-GFP) is enriched at ssu72∆ telomeres. Colocalization of Rad11-GFP with Taz1-mCherry, used as a telomere marker was performed in wt and ssu72∆ cells; n =3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM). More than 1000 cells were analyzed for each genotypeC) ssu72+ controls telomere length independently of rif1+. Epistasis analysis of telomere length of ssu72∆ and ssu72-C13S (catalytically inactive mutant) with rif1∆ was performed by Southern blotting of ApaI digested DNA using a telomeric probe. D) ssu72+ and stn1+ regulate telomere length in the same genetic pathway. Epistasis analysis of ssu72∆ and stn1-75 performed by Southern blotting of ApaI digested DNA using a telomeric probe. Two independently generated ssu72∆ stn1-75 double mutants are shown.
Figure 3A) Ssu72 is required for telomere recruitment of Stn1 in late S phase. ChIP analysis of stn1-myc in wt and ssu72∆ cells was performed in synchronized cdc25ts cells. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to ssu72+ control samples. Error bars represent standard error of the mean (SEM).B) 2D-gel analysis of NsiI telomeric fragments of wt and ssu72∆ strains. Smart ladder from Eurogentec was used for DNA size measurement.C) Serine 74 substitution to a phosphomimetic aspartate amino acid (stn1-S74D) is sufficient to confer ssu72∆ telomere defects. Telomere length epistasis analysis of ssu72∆ and stn1-S74D mutants were performed by Southern blotting of ApaI digested genomic DNA using a telomeric probe.E) Similar to ssu72∆ mutants, stn1-S74D is defective in telomere recruitment. ChIP analysis of stn1-myc and stn1-S74D-myc using a non-tagged strain as a control. n = 3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).
Figure 4A) ssu72+ and pol1+ regulate telomere length in the same genetic pathway. Epistasis analysis of ssu72∆ and pol1-13 mutants was performed by Southern blotting of ApaI digested DNA using a telomeric probe.B) Stn1-Pol1 interaction requires Ssu72 phosphatase. Immunoprecipitation experiments of Pol1-Flag with Stn1-Myc was preformed both in wt and ssu72∆ mutants. C) Immunoprecipitation of Ten1-Flag with Stn1-Myc in either wt or ssu72∆ was performed as control for Stn1-Ten1 complex integrity. D) Overexpression of polymerase alpha partially rescues telomere length defect of ssu72Δ mutants. Multi-copy vector with polymerase α under thiamine promoter were expressed both in wt or ssu72Δ cells.
Figure 5A) Telomere elongation of SSU72 down-regulated cells is telomerase dependent. Telomere Southern blot was carried out with HT1080 cells infected for 4 weeks with lentiviral particles carrying two independent shRNAs against SSU72 (CDS and UTR regions) and Luciferase (Luc) as control for shRNA. Knockdown efficiencies were determined by RT-qPCR using specific primers against hSSU72.B) Telomere elongation of SSU72 down-regulated cells is epistatic with STN1 downregulation. TRF analysis of HT1080 cells infected with the appropriate retroviral or lentiviral particles carrying either GFP shRNA, SSU72 shRNA (against UTR), STN1 shRNA or double SSU72 shRNA/STN1 shRNA. Cells were grown for 4 weeks and DNA was isolated to carry out Southern Blot analysis.C) hSSU72 down-regulation results in multi-telomeric signals (MTS) that are dependent on DNA replication. Visualization of mitotic spreads of HT1080 hSSU72 shRNA cells treated with aphidicolin and colcemid. Telomere FISH was carried out using a PNA-telomere probe. MTS are represented by arrows. Quantification of MTS per metaphase: n=4; **p ≤0.01 ****p ≤0.0001 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).
Figure 6A) hSSU72 downregulation results in telomere DNA damage foci (TIF). Cells with indicated shRNAs were fixed and IF-FISH analysis was performed using a 53BP1 antibody and a PNA-telomere probe. (Right) Quantification of cells with more than 5 telomeric 53BP1 foci: n=3; **p ≤0.01 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).B) hSSU72 is required for efficient loading of hSTN1 at human telomeres. ChIP was preformed using anti-FLAG antibodies and Southern blotting was performed using a human telomeric probe. Quantification of 3 independent ChIP experiments *p ≤0.05 **p ≤0.01 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).