EMBO/SourceData huggingface dataset:
Figure 2A Maximum intensity projections of fluorescent microscopy z-stacks of 5-day-old nematodes expressing the indicated transgenes are shown. White dashed lines outline the borders of muscle cells. Scale bar: 10 µm. The co-expression of HPI and HPII reduces the amount of α-Syn::YFP inclusions. B Quantification of α-Syn::YFP foci in 3- to 6-day-old animals. Product of mean foci fluorescence and foci area relative to muscle area is displayed (relFluoFoci). Data are shown as mean ± SEM (in %) (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. ** = p ≤ 0.01, *** p ≤ 0.001. C Motility Assay as a measure for transgene toxicity. Displayed is the mean number of body bends per 30 s ± SEM (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. ** p ≤ 0.01, *** p ≤ 0.001.D Maximum intensity projections of fluorescent microscopy z-stacks of 6-day-old animals expressing the indicated transgenes. Scale bar: 100 µm. The co-expression of HPI and HPII reduces the amount of Q35::YFP inclusions.E Quantification of Q35::YFP foci in WT and hsp-110 hairpin background. Shown is the mean ± SEM (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. * p ≤ 0.05, ** p ≤ 0.01.F Motility Assay as a measure for transgene toxicity. Displayed is the mean number of body bends per 30 s ± SEM (N = 3). Statistical analysis was done using one-way ANOVA with Dunnett's multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01.G Maximum intensity projections of fluorescent microscopy z-stacks of of 5-day-old nematodes expressing the indicated transgenes. White dashed lines outline the borders of muscle cells. Scale bar: 10 µm. M: muscle, H: hypodermis. Signal outside of muscle cells reveals spreading of α-Syn. H Quantification of animals showing α-Syn transmission at indicated ages. Displayed is the mean ± SEM (in %) (N = 4). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. *** p ≤ 0.001.
Figure 3A Maximum intensity projections of confocal microscopy z-stacks of 5-day-old animals expressing the indicated transgenes. White dashed lines outline the borders of muscle cells. Scale bar: 10 µm. The expression of the yeast SSE1N575Y E575A mutant cannot reverse the reduction of α-Syn::YFP foci in the hsp-110 hairpin expressing animals.B Quantification of motility as a measure for transgene toxicity. Displayed is the mean ± SEM (N = 3) amount of body bends/30s of animals expressing the indicated transgenes. Statistical analysis was done using two-way ANOVA and with Dunnett's multiple comparison test. n.s. = not significant, ** p ≤ 0.01, *** p ≤ 0.001. The presence of the yeast SSE1N575Y E575A mutant was not able to suppress the rescue of α-Syn::YFP toxicity in hsp-110 hairpin expressing animals.C Maximum intensity projections of fluorescent microscopy z-stacks of 5-day-old nematodes expressing the indicated transgenes. White dashed lines outline the borders of muscle cells. M: muscle, H: hypodermis. Scale bars: 10 µm. Signal outside of muscle cells reveals spreading of α-Syn.D Quantification of animals showing α-Syn transmission into the hypodermis at indicated ages. Displayed is the mean ± SEM (in %) (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, *** p ≤ 0.001.E Experimental set-up: Age-synchronized 4-day-old animals were subjected to heat stress for 3 h at 33°C (+HS) then returned to 20°C or left at 20°C (-HS), respectively. Arrows indicate imaging time points. The -HS controls were imaged at the same time point as the +HS animals after 24h at 20°C. Maximum intensity projections of fluorescent microscopy z-stacks of animals expressing the indicated transgenes. Scale bar: 20 µm. The impaired clearance of HS-induced FLUCSM::GFP foci in the hsp-110 hairpin expressing cells is rescued by co-expression of WT but not by the HSP-70 binding deficient SSE1N575Y E575A mutant.F Quantification of FLUCSM::GFP foci disaggregation. % disaggregation is calculated as 100 - ratio of FLUCSM::GFP foci area relative to total muscle area at the +HS 24h time point to the +HS 2h time point. Data are displayed as mean ± SEM (in %) (N = 3). Statistical analysis was done using one-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, ** p ≤ 0.01. All strains harbor the sid-1(pk3321) allele.
Figure 4A Maximum intensity projections of confocal z-stacks of 4-day-old animals expressing the indicated transgenes or harboring the indicated temperature-sensitive (ts) mutations. Muscle cells were stained with anti-myosin (green) and Alexa Flour 647-phalloidin (purple). Scale bar: 5 µm. Myosin misfolding reflects the exposure of the ts mutant phenotype of myosin(ts) [unc-54(e1301)] at the permissive temperate of 15°C by co-expression of the hsp-110 hairpins.B Quantification of motility as a measure for toxicity. Displayed is the mean ± SEM (N = 4) of body bends/30s of animals expressing the indicated transgenes or harboring the indicated mutations. Statistical analysis was done using one-way ANOVA with Dunnett's multiple comparison test. *** p ≤ 0.001. Co-expression of the hsp-110 hairpins exposed the ts mutant phenotype at the permissive temperate of 15°C, resulting in a significant increase in movement defects. All strains contain the sid-1(pk3321) allele.C Quantification of motility as a measure for transgene toxicity. Displayed is the mean ± SEM (N = 4) amount of body bends/30s of animals expressing the indicated transgenes or harboring the indicated mutations. Statistical analysis was done using two-way ANOVA with Tukey's multiple comparison test. * p ≤ 0.05. Expression of the hsp-110 hairpins caused movement defects with increasing age.D Maximum intensity projections of confocal z-stacks of 12-day-old animals expressing the indicated transgenes. Scale bar: 10 µm. FLUCSM forms foci with increasing age, which is markedly accelerated upon hsp-110 KD.E Quantification of animals with FLUCSM::GFP foci at indicated days of life. Data are displayed as mean ± SEM (in %) (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, ** p ≤ 0.01, *** p ≤ 0.001.F Quantification of motility as a measure for transgene toxicity. Displayed is the mean ± SEM (N = 3) amount of body bends/30s of animals expressing the indicated transgenes or harboring the indicated mutations. Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, *** p ≤ 0.001. Expression of the hsp-110 hairpins caused movement defects with increasing age. The beneficial effects of HSP-110 KD on amyloid toxicity is masked in older animals.G Quantification of motility as a measure for transgene toxicity. Displayed is the mean ± SEM (N = 3) amount of body bends/30s of animals expressing the indicated transgenes or harboring the indicated mutations. Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, , ** p ≤ 0.01, *** p ≤ 0.001. Expression of the hsp-110 hairpins caused movement defects with increasing age. The beneficial effects of HSP-110 KD on amyloid toxicity is masked in older animals. Data information: All strains harbor the sid-1(pk3321) allele.
Figure 1A) Left: Representative current traces from wildtype NavMs channels showing the first 0.1 s of 0.5 s activations from -180 mV holding potential recorded with the indicated extracellular cations. Right: Resulting current-voltage profiles profiles, where current magnitudes were normalized to the maximum inward current measured in the sodium condition.B) Left: Representative current traces from mutant channel E178D (as in A). Right: Resulting current-voltage profiles (error ± SEM; n = 6-9 for NavMs; n = 4-8 for E178D).
Figure 2A) Upper: Overview of the AB tetramer, with each of the monomers depicted in a different colour. The front monomer has been removed for clarity. The black box indicates the region expanded in the lower panel. Lower: An expanded view of the selectivity filter, showing only two monomers as pink and white ribbon cartoons with the selectivity filter residues drawn as sticks in the corresponding colours. Sodium ions are shown as cyan spheres and well-ordered waters as red spheres (at 0.4x their actual radii, for clarity). The final refined 2Fo-Fc electron density maps for the protein, ions and water are shown at 2.0 σ in dark grey mesh and 0.8 σ in light grey , and the initial difference map (without sodiums or waters included in the phase calculation) at 3.0 σ in green mesh.B) Superposition of the sodium ion binding sites found in the crystallographically-distinct AB and CD tetramers and the proposed binding sites from other studies. The tetramers were superposed with the LSQKAB program (Kabsch, 2010) from CCP4 (Winn et al., 2011) using residues 131-220 from all four monomers in each tetramer. Only two monomers from the AB tetramer are shown for clarity, and coloured as in panel A. Sodium ions from the AB tetramer are depicted as cyan spheres, those from the CD tetramer as teal spheres. Predicted sodium ion binding sites from Ulmschneider et al. (2013) are indicated as small black spheres and labelled on the right of the spheres according to the nomenclature (S0 to S4) in that study. Predicted sodium ion binding sites from Payendeh et al. (2011), based on a closed channel structure of the close orthologue NavAb, are indicated on the far right and labelled as per the nomenclature of that study (SiteHFS, SiteCEN and SiteIN). This and subsequent molecular figures were drawn with PyMol (The PyMOL Molecular Graphics System, Version 1.6, Schrödinger, LLC) unless otherwise stated.
Figure 3A) Top: Crystal structure overview of the wildtype AB tetramer (PDBID 5ZBZ), view as in Figure 2A.Left: Difference (Fo-Fc) electron density map (mutant minus wildtype) contoured at -2.5σ and drawn as red mesh, plotted on the structure of the wildtype (E178) protein, highlighting the consequences of the loss of the δ-carbon atom from residue 178 in the E178D mutant. For reference, the locations of the sodium ion sites present in the wildtype (E178) structure are depicted as cyan spheres. Both the structure and the ions are displayed as faded backgrounds so that the difference map can be seen more clearly.Right: Final refined 2Fo-Fc electron density map for the E178D mutant (PDBID 4X88), contoured at 1.5σ and shown as grey mesh, with the sodium ions depicted as cyan spheres.B) Surface representations of (left) wildtype NavMs pore and (right) the E178D mutant coloured by electrostatic charge calculated by APBS (Callenberg et al, 2010) and drawn with VMD (Humphrey et al., 1996), showing a slice through the middle of the tetramer. Sodium ions are shown as cyan spheres; only 2 are present in the mutant, instead of the 3 seen in the wildtype, with the top sodium missing in the mutant. The lower panels are expanded views of the selectivity filter regions, with the E178 side chains depicted in yellow sticks and the location of the main chain nitrogen of residue 179 indicated by the black circle. The alteration in the protein structure caused by removal of the methylene group of E178 exposes a positive region of the polypeptide backbone adjacent to the site where the top sodium ion is located in the wildtype protein; this likely contributes to why an equivalent sodium ion is not seen adjacent to this position in the mutant.
Figure 4Left: Sodium ions (cyan) in the selectivity filter of the NavMs sodium channel. Only two monomers (in white and pink) are shown for clarity. Coordinates are from the AB tetramer of PDBID 5BZB.Right: Potassium ions (blue) in the selectivity filter of the KcsA potassium channel. As for the NavMs structure, only two monomers (in grey and maroon) are shown for clarity. Coordinates are from PDBID 1K4C (Zhou et al., 2004).The two crystal structures were first superimposed using SSM (Krissinel and Henrick, 2004), and are drawn using the same scale, and with the correct ratio between the diameters of the ions used, so that the differences in the internal dimensions of their selectivity filters can be seen, thus making it clear how much more space there is available in the NavMs SF for intervening water molecules. In the KcsA channel, the ions are directly coordinated to the polypeptide backbone atoms, whereas in the NavMs SF, the closest atoms (side chains) are obviously at longer distances than direct bonding to the ions, enabling interactions via water molecules.
Figure 1C Upper panel: senescence curve of a representative est2∆ clone. DNA samples were prepared at the indicated time points to analyze telomere length (middle panel) and t-circles production (lower panel and graph) during senescence and cell immortalization. Telomeres were analyzed by Southern blot of the XhoI cut DNA hybridized with a TG1-3 probe. The presence of t-circles was monitored by RCA and analyzed by TG1-3 probed dot blot. The graph shows signal intensities of the dots measured with the Fiji software (arbitrary units). Similar results were obtained with at least 16 other est2∆ clones.E) Dot blot of the RCA product either treated or not with the Exonuclease VII that degrades linear single-stranded DNA in both the 3' to 5' and 5' to 3' directions (upper panel). The membrane was hybridized with a telomeric TG1-3 probe. The signal intensities of the dots are shown (lower graph).
Figure 2A Senescence curve of a representative est2∆ clone and corresponding dot blot of the RCA product hybridized with the indicated subtelomeric or telomeric probes. t-circle amounts were evaluated based on signal intensity of the dots and were expressed as fold increase over the signal in the absence of Φ29 (lower graph).B Senescence curve of a representative est2∆ clone (upper panel). DNA samples were prepared at the indicated time points to analyze telomere length by Southern blot (middle panel) and t-circles production by RCA assay performed in the absence of dCTP and dATP.(lower panel). The graph shows signal intensities of the dots measured with the Fiji software (arbitrary units).
Figure 3C Detection of the t-circles co-immunoprecipitated with Nic96-RFP from the extracts obtained from several type II survivors. The DNA co-immunoprecipitated with Nic96-RFP was amplified by RCA assay, loaded on a dot blot and detected with a telomeric TG1-3 probe. The numbers above the blot refer to the growth curves in (A) and correspond to type II survivors produced in independent cultures. The lower panel shows the efficiency of Nic96-RFP IP that was evaluated by western blot with anti-mCherry antibody.D Quantification of the RCA product after co-IP of the C-circles with Nic96-RFP showing the effect of ADA2 deletion. Dot signal intensities were quantified using the Fiji software and were calculated as the percentage of INPUT that was immunoprecipitated normalized to est2∆ signals. Relative signals were calculated by setting the mean of est2∆ signals from each independent experiment to 1. Means and s.e.m. of the six independent co-IPs experiments each with 2-6 clones per genotype are shown. Outliers were removed using ROUT robust outlier test. The p-values from one-way ANOVA were adjusted using the Bonferroni's correction for multiple comparisons. est2∆ versus est2∆ NIC96-RFP (p=0,000007), est2∆ NIC96-RFP versus est2∆ ada2∆ NIC96-RFP (p=0,00012).***P<0,0001.E Quantification of the RCA product after co-IP of the C-circles with Nic96-RFP showing the effect of SUS1 deletion. The graph shows means and s.e.m. of the three independent co-IPs with 2-5 clones per genotype. Outliers were removed using ROUT robust outlier test. The p-values from one-way ANOVA were were adjusted using the Bonferroni's correction for multiple comparisons. est2∆ versus est2∆ NIC96-RFP (p=0,002), est2∆ versus est2∆ sus1∆ NIC96-RFP (p= 0,0006). **P<0,001, ***P<0,0001.
Figure 4A Senescence curves of two est2∆ and two est2∆ ADA2-Myc clones used to generate type II survivors for Co-IP-RCA. This experiment is representative out of the 4 independent experiments performed during this study.C Detection of the C-circles co-immunoprecipitated with Ada2-Myc in type II survivors. The DNA co-immunoprecipitated with Ada2-Myc was amplified by RCA, loaded on a dot blot and detected with a telomeric TG1-3 probe. The numbers above the blot refer to clones in (A) and correspond to type II survivors produced in independent cultures. The lower panel shows the efficiency of Ada2-Myc IP analyzed by western blot with anti-Myc antibody.
Figure 5A Mean senescence profiles of the est2∆ (n=7) and est2∆ sus1∆ (n=8) clones isolated from the same heterozygous diploid. The error bars are SDs. B Mean senescence profiles of the est2∆ (n=10) and est2∆ sac3∆ (n=10) clones isolated from one heterozygous diploid. The error bars are SDs.
Figure 6A Mean senescence profiles of the est2∆ (n=10) and est2∆ shs1∆ (n=16) clones. The error bars are SDs. B Mean senescence profiles of the est1∆ (n=6) and est1∆ bud6∆ (n=7) clones. Est1, similarly to Est2, is essential for telomere maintenance. est1∆ has been used in this experiment instead of est2∆ because EST2 and BUD6 genes are linked on chromosome XII. The error bars are SDs.
Figure 1Results of three independent microscopy-based screens in U2OS cells. Knockdown of Luciferase (blue) and the positive controls PPM1D and βTrCP (red) are indicated. A low/high Z-score indicates less/more phospho-histone H3 positive cells compared to the control, reflecting reduced/increased numbers of cells going into mitosis in the respective knockdowns after 5 Gy of IR. Deconvolution of the 41 primary hits in U2OS cells by four separate siRNA oligonucleotides. Grey represents a recovery value that is similar to the Luciferase control (<0.5 SD) and the red represents an altered recovery value (>0.5 SD) in two independent experiments.U2OS cells (D) and RPE1 hTert immortalized primary epithelial cells (E) were transfected with siRNA oligos targeting the hits in a similar setup as described in A), but now analyzed for mitotic entry by MPM2 and PI staining by flow cytometry. For normalization see materials and methods. Error bars represent the SEM of three independent experiments.
Figure 2RPE1 cells were depleted for p53 in combination with the indicated proteins by siRNA, in conditions used for the primary screen (2 Gy of IR). Mitotic entry was analyzed by MPM2 and PI staining by flow cytometry. For normalization see materials and methods. Error bars represent the SEM of three independent experiments.U2OS cells were transfected with the indicated siRNA oligonucleotides in a setup similar to the primary screen, but with 10 Gy of IR and in the presence of ATM and ATR inhibitors. Mitotic entry was analyzed by MPM2 and PI staining by flow cytometry. For normalization see materials and methods. Error bars represent the SEM of three independent experiments.U2OS cells were transfected with the indicated siRNA oligos using the protocol of the primary screen and fixed for immunofluorescence after 0, 2 and 24 h after 5 Gy IR. Samples were stained with antibodies for γH2AX (D) or 53BP1 (E) and analyzed through automated confocal microscopy and specifically developed image analysis software. From one experiment two to four separate images with a total of at least 100 cells were analyzed for each knockdown. Shown are the number of γH2AX (D) and 53BP1 (E) foci per cell. In the box plots, the median with the first and third quartile is indicated, and the whiskers are drawn down to the 10th percentile and up to the 90th.
Figure 3U2OS cells were transfected with four different siRNA oligonucleotides targeting PHF6, synchronized in G2 and subsequent recovery after 5 Gy of IR and the addition of nocodazole was analyzed by flow cytometry with MPM2 staining PPM1D and βTrCP were used as positive controls.U2OS cells were transfected with four different siRNA oligonucleotides targeting PHF6, synchronized in G2 or cells were lysed and analyzed using western blotting with the indicated antibodiesU2OS WT, PHF6 knockout and PHF6 knock out cells that were reconstituted with GFP-PHF6 were synchronized in G2 and recovery was determined after 2 Gy of IR using flow cytometry with pHH3 stainingU2OS WT, PHF6 knockout and PHF6 knock out cells that were reconstituted with GFP-PHF6 were synchronized in G2 cells were lysed and analyzed using western blotting with the indicated antibodiesU2OS WT and PHF6 knockout cells were irradiated using 3 Gy of IR and cells were fixed at different time points for immunofluorescence. In (F) is presented 53BP1 IRIF intensity. In (G) is shown the number of γH2AX foci per cell.U2OS WT and PHF6 knockout cells were left untreated or irradiated with 3 Gy and processed after 1 h for comet assay analysis. The comet tail moment was analyzed in at least 50 individual cells.
Figure 4U2OS cells stably expressing GFP-PHF6 and mCherry-NBS1 were laser-irradiated and cells were analyzed by time-lapse imaging.U2OS cells stably expressing GFP-PHF6 were laser-irradiated and cells were analyzed by time-lapse imaging. In (B), the PARP inhibitor Olaparib was added 30 min before laser-induced damage. At least 60 cells were analyzed in three individual experiments and error bars represent the SD (lower panel). Scale bar is 5 μM.GC92 SV40 immortalized human fibroblasts containing the I-SceI NHEJ reporter were transfected with the indicated siRNA oligos and I-SceI. 48 h later the cells were fixed and analyzed by flow cytometry. Represented is the relative repair efficiency as compared to the Luciferase control.Clonogenic survival assays of U2OS WT and PHF6 knockout cells that were depleted for luciferase or XRCC4 and treated with 2, 3 or 4 Gy IR. Shown is the relative survival as compared to the undamaged control. Error bars represent the SEM of three individual experiments.GC92 SV40 immortalized human fibroblasts containing the I-SceI NHEJ reporter were transfected with the indicated siRNA oligos and I-SceI. 48 h later genomic DNA was extracted and repair of I-SceI cut-sites analyzed through junction analysis (n= independently derived sequences, see materials and methods for details).
Figure 1A. RGFP966 (966)-dependent HDAC3 inhibition resulted in significant and robust neurite outgrowth (24h in culture) compared to HDAC class II, HDAC1/2 and HDAC1 or 3 inhibitions via 69A, 963 and 233 respectively. Data information: Data expressed as mean fold change N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle ANOVA followed by Bonferroni test).B, C. Specific pharmacological inhibition of HDAC3 facilitates DRG neurite outgrowth (average neurite outgrowth after 24h in culture, Tuj1 positive neurons) on PDL-laminin or Myelin substrates. Scale bars, 100 μm. Data information: Data expressed as average neurite length ± s.e.m. N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle Student's t-test)D, E. Immunoblotting shows significantly increased H3K9 acetylation after 966 vs vehicle. Data information: Data expressed as mean fold change N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle Student's t-test)F, G. AAV-mediated mutant HDAC3 (Y298H, V5) infection of DRG neurons induces neurite outgrowth (Tuj-1/V5 double positive neurons) after 36h in culture, which is repressed by wildtype HDAC3 overexpression. Scale bars, 100 μm. Data information: Data expressed as average neurite length ± s.e.m. N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus V5 vs AAV-GFP ANOVA followed by Bonferroni test).