EMBO/sd-character-level-ner huggingface dataset:
Figure 3(A) The formation of secondary ParBF-DNA complexes requires the box II motif. EMSA were performed with a 144-bp 32P-labelled DNA fragments (C144) carrying a single 16-bp parS binding motif. Reaction mixtures containing 100 µg.ml-1 sonicated salmon sperm DNA were incubated in the absence (-) or the presence of increasing concentrations (grey triangle; 10, 30, 100, 300, and 1000 nM) of ParBF or ParBF-3R*. Positions of free and bound probes are indicated on the left. B1 represents complexes involving the specific interaction on the 16-bp binding site, while B"2 and B"3 complexes represent secondary complexes involving the parSF site with one or two additional nsDNA-binding interactions, respectively (Sanchez et al, (B) ParBF cluster formation requires the box II motif. Epifluorescence microscopy of ParBF-3R*-mVenus from DLT3566 is displayed as in Fig. 1D. White bars: 1 µm (C) ParBF in vivo DNA binding in the vicinity of parSF sites requires the box II motif. ChIP-seq was performed on DLT3726 carrying parSF in the xylE chromosomal locus and expressing ParBF-3R* variant. ParBF-3R* DNA binding profile displayed the number of nucleotide reads as a function of the E. coli genomic coordinates. The peak at parSF covered approximately 950-bp, which corresponds to the 402-bp between the 1st and 10th specific binding sites and ~280-bp on each sides (representing the average size of the DNA library; see Appendix Fig. S3E). No ParBF-3R* enrichment was found on parSF-flanking DNA and elsewhere on the chromosome. Inset, zoom in on the right side of parSF over 5-Kbp with the ParB density, normalized to 1 at the first bp after the last parS binding repeat, plotted as a function of the distance from parSF. Note that a highly similar DNA binding pattern is obtained with ParBF -3R*-mVenus (strain DLT3566; Appendix Fig. S3C)
Figure 4Wildtype SIRT7 or its RF mutant was re-expressed in Sirt7-knockdown MEF cells (A) Wildtype SIRT7, but not its RF mutant, restores mitochondria mas in Sirt7-deficient MEF cells Mitochondria mass was determined by Mito Tracker Red staining (B) (mean ± SD, n=3 experimental replicates, *p<0.05; **p<0.01; n.s.=not significant, unpaired two-tailed t test) Wildtype SIRT7, but not its RF mutant, restore ATP leve in Sirt7-deficient MEF cells Cellular ATP was quantified and normalized to cell number (C) (mean ± SD, n=3 experimental replicates, *p<0.05; **p<0.01; n.s.=not significant, unpaired two-tailed t test) Wildtype SIRT7, but not its RF mutant, restore cellular respiration in Sirt7-deficient MEF cells Respiration flux was determined as indicated (D). (mean ± SD, n=3 experimental replicates, *p<0.05; **p<0.01; n.s.=not significant unpaired two-tailed t test) Wildtype SIRT7, but not its RF mutant, downregulates SIRT7 target genes involved in mitochondria biogenesis. Wildtype SIRT7 and its RF mutant were stably expressed in MEF cells (E) Wildtype SIRT7, but not its RF mutant, downregulates SIRT7 target genes involved in mitochondria biogenesis SIRT7 target gene expression was determined and presented as a heatmap on a log2 scale (F) Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs. The H3K18ac level at the promoter of SIRT7 target genes (Gfm2, Mrps31, Mrps33, and Mrpl40) was determined by ChIP-qPCR, Rabbit IgG was used as a negative control (G) ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t test Inhibition of Prmt6 decreases mitochondria mas of control cells, but not Sirt7-knockdown MEF cells Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Mitochondria mass was determined by Mito Tracker Red staining (H) ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t test Inhibition of Prmt6 decrease ATP leve of control cells, but not Sirt7-knockdown MEF cells. Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Cellular ATP was quantified and normalized to cell number (I) ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t test Inhibition of Prmt6 decrease the respiration flux of control cells, but not Sirt7-knockdown MEF cells. Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Respiration flux was determined as indicated (J). ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t test
Fig.Figure 5Number of Us11-GFP expressing P6 SCG neurons infected with HSV-1 following IFNα treatment for 18 hours prior to infection or for 18 hours at 3 days prior to infection. n=9 biological replicates. Percent colocalization of vDNA foci detected by click chemistry to PML at 8 dpi in SCG neurons infected with HSV-1EdC following IFNα treatment for 18 hours prior to infection or for 18 hours at 3 days prior to infection. Each point represents the percentage of 20 vDNA foci that colocalized to PML from 3 biological replicates. Quantification of PML puncta at time of infection in P6 SCG neurons treated with IFNα (600 IU/ml) for 18 hours prior to infection or for 18hours at 3 days prior to infection. n=60 cells from 3 biological replicates. Percent colocalization of vDNA foci detected by click chemistry to PML at 3 dpi in SCG neurons with HSV-1EdC infected with ICP0-null mutant HSV-1, n212, or a rescued HSV-1 virus, n212R, in P6 SCG neurons treated with IFNα for 18 hours prior to infection or for 18hours at 3 days prior to infection. Each point represents the percentage of 20 vDNA foci that colocalized to PML from 3 biological replicates.
Figure 4(A, B) U2OS 2-6-3 parental (WT) or RAP80-/- cell lines were transfected with siRNAs targeting BRCA1 followed by nucleofection of the indicated GFP fusion proteins. 48 h post-nucleofection, mCherry-LacR-FokI expression was induced for 5 h prior to being processed for fluorescence microscopy for GFP and mCherry. Shown in (A) is the quantitation of GFP fluorescence at the mCherry focus where the bars represent the mean ± S.D (n= 50, 50, 40, 40, 40, 40, 20, 20, 30, 30 cells analyzed from 3 biological replicates). Representative micrographs for the BRCA1 and BRCA1-I26A conditions are shown in (B). Additional micrographs for the other conditions are in Figure EV2C. (C) RPE-1 hTERT p53-/- BRCA1-/- Cas9 cells (WT) or their isogenic RAP80-/- counterparts expressing the indicated GFP fusion proteins were processed 1 h post-irradiation (10 Gy) for immunofluorescence using GFP and antibodies against BRCA1 and γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D (n=3 biological replicates). Representative micrographs are shown in Figure EV3B. (D) RPE-1 hTERT p53-/- BRCA1-/- Cas9 cells or their isogenic RAP80-/- counterparts expressing the indicated GFP fusion proteins were processed 1 h post-irradiation (10 Gy) for immunofluorescence using GFP and antibodies against RAD51 and γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D (n=3 biological replicates). Representative micrographs are shown in Figure EV3C. Scale bar is 5 μm in (B).
Axon tracts in the corpus callosum (CC) of the Plcg1f/f;Nes-Cre mice are more diffuse than those of control mice.Figure 5(A) Cumulative histograms based on fractional anisotropy (FA) values. (B), show the CC volume relative to FA values. As FA values increased, the structural (CC volume differences increased between the control and Plcg1f/f;Nes-Cre mice. (n = 4; Shapiro-Wilk and t-test; *p < 0.05). Data are presented as the mean ± s.e.m. (C), Cumulative histograms based on fractional anisotropy (FA) values. (D), show the number of tensors relative to FA values. As FA values increased, the structural number of tensors) differences increased between the control and Plcg1f/f;Nes-Cre mice. (n = 4; Shapiro-Wilk and t-test; *p < 0.05). Data are presented as the mean ± s.e.m. (E) Cumulative histograms based on fractional anisotropy (FA) values. (F) show the CC volume and the number of tensors relative to FA values. As FA values increased, the structural (CC volume and the number of tensors) differences increased between the control and Plcg1f/f;Nes-Cre mice. Depending on the number of tensors, the length in the CC appeared to be shortened (E, F). (n = 4; Shapiro-Wilk and t-test; *p < 0.05). Data are presented as the mean ± s.e.m.
Figure 6A) Representative stereoscopic images and Western blot analysis of IκBα KO organoids infected with the indicated sh-RNA for NF-κB elements. Data information Scale bars in A, 100 μm, (B) qPCR analysis of the indicated ISC and differentiation genes in the different sh-RNA treated organoids. The canonical NF-κB target gene Cxcl10 is shown as positive control of the experiment. Data information 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant. (C) Representative stereoscopic images of IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ-6438. Data information Scale bars in 50 μm. (D-E) qPCR analysis of the indicated adult and fetal ISC genes (D) and elements of the Notch and IFN pathway (E) (differentially expressed in IκBα KO organoids) in IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ-6438. Data information: 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant. (F) Representative stereoscopic images of IκBα KO organoids treated with dexamethasone as IFN inhibitor and DAPT as Notch inhibitor. Data information Scale bars in F, 50 μm. (G) qPCR analysis of IκBα KO organoids untreated or treated with dexamethasone and DAPT. Data information: 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant.
Figure 3(A) Schematics of the RNAi screen strategy. Females expressing UASPoloT182D under the control of eyless-Gal4 promoter were crossed with males carrying a specific UASRNAi transgene. Crosses were left either at 18°C or 25°C and adult offspring viability was assessed. (B) Representative images of the eye phenotype observed in adult flies that were expressing UASPoloT182D in the eye imaginal disc during development at indicated temperatures. An example of an eye from an adult fly co-expressing UASPoloT182D with UASRodRNAi is also shown. Expression of UASlacZ transgene in the eye imaginal disc was used as control for overexpression and UAS dilution effect. (C) Graph represents the mean percentage of males born at 25°C either expressing eyGal4-driven UASPoloT182D together with UASlacZ or in combination with UASRodRNAi (n≥3 independent crosses for each condition). The number of males with the genotype of interest born in each cross was normalized to the mean total number of males that are born in a control cross (eyGal4>UASlacZ, n=9 independent crosses). Data information: Data are shown as mean ± SD. Scale bar: 5 μm. (D) Selected stills from live cell imaging of neuroblasts expressing either UASPoloT182D alone or in Rod-depleted background (UASPoloT182D+UASRodRNAi). Neuroblasts without transgene expression were used as control and neuroblasts expressing UASPoloT182D together with UASlacZ were used as control for UAS dilution effect. (E) Quantification of the time spent in prometaphase (from NEBD until last KT alignment at the metaphase plate) for neuroblasts shown in (D) (n≥17 neuroblasts for each condition, n≥4 independent experiments). Statistical analysis was calculated using an one-way ANOVA test for multiple comparisons. p values: ns, not significant; **, <0.01. Data information: Data are shown as mean ± SD. Scale bar: 5 μm. (F) Quantification of the percentage of prometaphase/metaphase mitotic figures per brain showing abnormal chromosome number (>8 chromosomes (chr) (n≥360 neuroblasts from at least 7 larvae brains for each condition, n≥3 independent experiments). Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ns, not significant; **, <0.01. Data information: Data are shown as mean ± SD. Scale bar: 5 μm.
In vivo, a unique phosphorylation of the Phospho-FFAT motif allows STARD3 and VAP-A/VAP-B complex formation and the establishment of ER-endosome contacts.Figure 4A,B: Binding assays. Immunoprecipitation (GFP-Trap) experiments between GFP-tagged VAP-A (WT and KD/MD mutant; A), VAP-B (B) and Flag-tagged STARD3 (WT and S209A, S209D and S209D/P210A mutants). Please note that the mutations modify the FFAT and Phospho-FFAT scores of this region of STARD3: compared to WT STARD3 (Phospho-FFAT: 2.5; conventional FFAT: 6.5), the STARD3 S209A, mutant has both low Phospho-FFAT and FFAT scores (6.5), the S209D mutant has a high conventional FFAT score (2.5) but a low Phospho-FFAT score (6.5), and the double mutant has a conventional FFAT score of 1.5 and a Phospho-FFAT score of 5.5. Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag, anti-GFP and anti-Actin antibodies. Immunoprecipitated material was analyzed using anti-Flag and anti-GFP antibodies. Recruitment assays. GFP-VAP-A (C ; green) were left untransfected (C) and labelled using anti-Flag (magenta) antibodies. The subpanels on the right are higher magnification (3.5×) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Scale bars: 10 µm. Inset scale bars: 2 µm. : Recruitment assays. GFP-VAP-A D, E, G; green) and GFP-VAP-A KD/MD-expressing cells (F; green) were transfected with Flag-STARD3 (D, F), Flag-STARD3 S209A (E), and Flag-STARD3 S209D/P210A (G), and labelled using anti-Flag (magenta) antibodies. The subpanels on the right are higher magnification (3.5×) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Scale bars: 10 µm. Inset scale bars: 2 µm. H: Pearson's correlation coefficients between VAP-A (WT or KD/MD mutant) and STARD3 (WT or S209A, S209D, S209D/P210A, FA/YA mutants) staining are shown. Each dot represents a single cell (number of cells: VAP-A-STARD3: 22; VAP-A-STARD3 S209A: 25; VAP-A-STARD3 S209D: 27; VAP-A-STARD3 S209D/P210A: 26; VAP-A-STARD3 FA/YA: 20; VAP-A KD/MD-STARD3: 20, from at least three independent experiments). Means and error bars (SD) are shown. Kruskal-Wallis with Dunn's multiple comparison test (**P < 0.01; ***P < 0.001).
Figure 4(A) ATPCL mutant male flies show reduced ATPCL activity at their 4th week. N=7 per group. (B) Non-targeted metabolome reveals 71 singinifcatly altered metabolites in young wild-type versus young +/atpcl mutants, while 439 metabolites are altered in the midlife groups out of 1,658 metabolite compounds. N=6 per group. (C) Targeted metabolite measurements show decreased acetyl-CoA and succinate in midlife +/atpcl mutants. N=4-6 per measurement. (D) Quantification of histone H3 and H4 acetylation in +/atpcl mutants at 4 weeks old (midlife). Age-associated hyper-acetylated histone states are reduced in +/atpcl mutant flies (H4K12, H3K14, H3K9/14 and H3K23). N=5-6 per group. (E) +/atpcl male flies show an increased mean lifespan and slower mortality rates in midlife. Survival 90% control = 26 days, +/atpcl = 30 days, Median survival for control = 38 days, +/atpcl = 50 days. N=220 (control), and 202 (+/atpcl). Log Rank Test, χ2 =67.87, p<0.0001. Error bars indicate the S.E.M. in all the graphs. Unpaired two tailed t-tests were used for calculating the p-values in A-D. All male flies were collected from a mixed male/female population.
Figure 7A Functional NEAT1_2 RNA domains for paraspeckle assembly and shell-localization of the 5′ and 3′ ends of the NEAT1_2 within the paraspeckles. Summary of the NEAT1_2 organization within the paraspeckle and the size of the paraspeckles in the representative NEAT1_2 mutants used in this study. B The triblock copolymer micelle model for paraspeckles. NEAT1_2 RNPs are treated as ABC triblock copolymers. Putative RNA-binding proteins (blue circles) interact with the shell-forming domains and the proteins involved in the paraspeckle assembly (e.g., NONO) (gray circles) mainly bind to the middle assembly domain. The NEAT1_2 RNPs are microphase-separated into paraspeckles as block copolymer micelles to form spherical and cylindrical shapes. The length of the blocks and the transcription rate of NEAT1_2 influence the organization, size, and shape of the paraspeckles.
Figure 4A. Plx1 and PP2A-B56 competes for Apc1-loop500. MBP-fused Apc1-loop500 WT or its derivatives (∆11, deletion of 11 residues including the B56-binding motif) was incubated with the 35S-labelled Flag-B56γ in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated with indicated amounts of WT Plx1-PBD or Plx1-PBD Pincer mutant (Pin) for 15 min. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or Coomassie brilliant blue (CBB) staining. B. Dephosphorylation of T532, T539 and S558 on Apc1 by PP2A-B56. MBP-fused Apc1-loop500 WT or its derivatives (L557A/V560A and E562A) was incubated in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, isolated by amylose beads and further incubated in the presence (78 nM) or absence of purified PP2A-B56γ at 23˚C for 20 min. The proteins were recovered by amylose beads, separated by SDS-PAGE and detected by immunoblotting with phospho-specific or MBP antibodies. The signals of a phospho-specific antibody and MBP antibody on the same membrane were detected in the 800- and 700-nm channels, respectively. The two channels were pseudo-coloured (green; pT532, pT539 and pS558, red; MBP) and overlaid in the bottom panel (Overlay). The signal of pS558 was very faint in L557A/V560A mutant probably because these two residue are close to S558, interfering with pS558 antibody recognition. C. Dephosphorylation of Apc1-loop500 by PP2A-B56 in the presence of Plx1-PBD. MBP-fused Apc1-loop500 WT or its derivatives (L557A/V560A or E562A) was incubated in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated with 10 µg of WT Plx1-PBD for 15 min. The complexes were isolated by amylose beads and incubated in the presence (78 nM) or absence of purified PP2A- B56γ at 23˚C for 20 min. The proteins were recovered by amylose beads, and analysed as described in (B).
a) Time-course experiment of HeLa cells transfected with Flag–Parkin wild-type and treated with CCCP for the indicated periods.figf6(a) Time-course experiment of HeLa cells transfected with Flag-Parkin wild-type and treated with CCCP for the indicated periods. Total lysates were prepared, and western blots were probed with specific anti-Parkin, anti-PINK1, anti-TOM20 and anti-VDAC1 antibodies. β-Actin probing served as a loading control. All of the proteins detected are endogenously expressed, except Flag-Parkin. (b) Pathogenic Parkin mutants fail to poly-ubiquitylate endogenous VDAC1 in response to CCCP treatment. HeLa cells were transfected with empty vector (-), Flag-Parkin wild-type or pathogenic mutants along with 6×His-tagged ubiquitin wild-type for 24 h. Cells were treated with CCCP for 6 h and subsequently protein lysates were prepared in urea buffer. Denaturing conditions of Ni-NTA affinity purifications were used to specifically pulldown only proteins covalently modified with 6xHis-tagged ubiquitin. Western blots of protein lysates and purified Ni-NTA agarose elutions were probed with anti-VDAC1, anti-TOM20 and anti-Parkin antibodies. Modification of VDAC1 was observed in cells expressing Parkin wild-type and the functional mutant G328E, whereas non-functional mutants did not have this effect. ( (c) In response to CCCP treatment, Parkin catalyses predominantly K27-linked poly-ubiquitin chains on endogenous VDAC1 and is itself strongly poly-ubiquitylated. HeLa cells were transfected with an empty vector (-) or Flag-Parkin wild-type along with 6×His-tagged ubiquitin variants for 24 h. Treatment of cells, preparation of lysates and Ni-NTA purification were performed as described in c and probed for Parkin, VDAC1 and β-actin. WT, wild-type; Ub, ubiquitin.
Mathematical model explains average number and length of microtubules at different tubulin expression levels A.Figure 6A cartoon depicting four possible states of a microtubule nucleation site considered in the mathematical model, namely unbound, bound, nucleating and microtubule growing, as well as the transition between them. The transition rates depend on the dimer concentration c. An unbound nucleation site transitions to a bound state when a tubulin dimer binds to it with rate kmcp. A bound nucleation site starts nucleation with rate kncn and adds tubulin dimer sequentially with rate kmc to grow the microtubule. Here p and n (where p+n=12 being the size of the critical nucleus, see Methods) represents the cooperativity of tubulin dimer binding to a free and a bound nucleation site, respectively. The minimum value of MSE in MT number (shown by black points) increases with the increase in binding cooperativity (in curves from left to right). The MSE in MT length at binding rates corresponding to MSE minimum in MT number (shown by open black circles) decreases with increase in binding cooperativity. The parameter set shown by star symbol is selected for comparing the modelling results with the experiments. The x-values are multiplied by a factor of (10 − 5 )p to present the plots in a common range for easy comparisons. The microtubule number increases with the increase in the concentration of tubulin dimers. Microtubule number scales with tubulin concentration at low expression. At high expression, microtubule number is limited by the number of nucleation sites. Shown are mean +/- standard deviation from all determined values. The average microtubule length remains close to the maximum length 6 μm. The circles are obtained by averaging results from 200 stochastic simulations of the model equations and stars are the experimental data (blue stars are for (α2+, α2++, α2+++) strains). The error bar shows the standard deviation in the data and simulation results. For the WT expression level (c0 = 36.6 (m0+p+n)), the rate of nucleation is faster than the microtubule growth. Then the microtubule number is determined by the number of nucleation sites. The average microtubule length is around 1 μm (shown by dashed arrow) when the last nucleus forms. At intermediate tubulin numbers (c0 = 16 (m0+p+n)), corresponding to the α1c-term mutant, the microtubule formation rate is comparable to nucleation rate. The average microtubule length is around 4 μm (shown by dashed arrow) when the last nucleus forms.