EMBO/sd-character-level-ner huggingface dataset:
Production of telomeric circles during senescence and survivor formation in S.Figure 1A Schematic of the RCA assay. The Φ29 polymerase produces long telomeric single strand concatemers of the circular DNA in a self-primed reaction. B Schematic of the senescence assay. Senescence assays were performed starting from the spores isolated from the heterozygous telomerase-negative diploids. The spore colonies were inoculated at OD600=0.01 in 15 ml liquid YPD medium. Every 24 hours, the cell density was measured and a new culture was restarted at OD600=0.01. Samples were collected at each time-point for further analyses. C 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. D RCA products were resolved by denaturing agarose gel electrophoresis, transferred on a membrane and hybridized with a telomeric TG1-3 probe. 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 1A. Protein phase separation can lead to the formation of protein assemblies with varying dynamics. Soluble proteins may phase separate into liquid-like droplets or insoluble deposits that according to their viscoelastic properties and their ability to exchange components with the cytoplasm vary from being dynamic to static. B. Design of the modular system to measure the effect of protein phase separation on cell fitness. C. Growth media composition and the essential/non-essential/toxic roles of Ura3p. D. The model proteins consist of a fusion between Ura3p and GFP (URA3sol, left) and the amyloid-β-peptide of 42 residues (Aβ) (URA3agg, right). After 18 hours of expression Ura3psol remains homogeneously distributed whereas Ura3pagg is accumulated into intracellular foci.
Figure 2(A) Scheme of the experiment. BALB/c mice were depleted or not of CD8+ T cells 3 days before the injection of 7x104 MM cells and subsequently treated with either 800 μg BoxA (n =9) or PBS (n =7) 3 times a week, for 10 times in total. Every week mice were surveyed by BLI. (B) Representation of the BLI of the four groups of mice. Left panel: CD8+ T cell depleted mice treated with BoxA (n=9) or PBS (n=7). Right panel: non-depleted mice treated with BoxA (n=11) or PBS (n=7). (C) Kaplan-Meier survival curves of the 4 groups of mice shown in panel B. Statistics: Gehan-Breslow-Wilcoxon test.
Figure 4Primary hippocampal neurons were transduced with GFP or GA175-GFP after 4 days in vitro, incubated for 7 days (DIV 4+7), and treated with vehicle (DMSO), MG132 (10µM) or rolipram (30 µM) for 16h. (A) Immunofluorescence reveals enhanced cytoplasmic TDP-43 levels in neurons with poly-GA aggregates or treated with MG132. Arrows mark punctate TDP-43 staining. Rolipram treatment reduced cytoplasmic TDP-43 in GA175-GFP neurons. Scale bar denotes 20 µm. (B) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced neurons. n=4 biological replicates. In total 462 GFP and 371 GA175-GFP cells treated with vehicle, and 386 GFP and 529 GA175-GFP cells treated with MG132, 513 GFP and 434 GA175-GFP cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. Primary hippocampal neurons were transduced with GFP or GA175-GFP after 4 days in vitro, incubated for 7 days (DIV 4+7), and treated with vehicle (DMSO), MG132 (10µM) or rolipram (30 µM) for 16h. (C) Immunoblot to show effects of MG132 and rolipram on GA175-GFP and GFP expression. Primary hippocampal neurons were transduced with GFP or GA175-GFP after 4 days in vitro, incubated for 7 days (DIV 4+7), and treated with vehicle (DMSO), MG132 (10µM) or rolipram (30 µM) for 16h. Filter trap assay with quantification of SDS-insoluble aggregated GA175-GFP. n=5 biological replicates. Scatter plot with mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. (F,G) HeLa cells were co-transfected with RFP-TDP-CTF and GFP or GA175-GFP for 2 days. For the final 16h, cells were treated with rolipram (30 µM) or MG132 (10 µM). Filter trap assay of SDS-insoluble TDP-CTF aggregates quantified by densitometry. n=4 biological replicates. Scatter dot plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. (H,I) HeLa cells were co-transfected with TDP-43ΔNLS-GFP and iRFP or GA175-iRFP for 2 days. For the final 16h, cells were treated with either vehicle or rolipram (30 µM). Filter trap assay of SDS-insoluble TDP-43ΔNLS-GFP aggregates quantified by densitometry. n=3 biological replicates. Scatter dot plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.
Mitochondrial dynamics in Sept2 depleted cells.Figure 2(A) Live cell imaging of mock treated or Sept2 depleted U2OS cells analyzed with the mito-PA-GFP mitochondrial fusion assay. Mito-PA-GFP is photoactivated in the indicated ROI and the decrease in fluorescence is followed in the same ROI, correcting for cell movement (B) Quantification of mitochondrial fusion rates in mock treated or Sept2 depleted cells expressed as a means of three independent experiments SEM. (C) Quantification of fission rates in mock and Sept2 depleted cells. n=116 fission events for Sept2 siRNA, n=83 fission events for mock, two independent experiments. (D) Live cell imaging of U2OS cells stably transfected with a GFP targeted to the mitochondrial outer membrane (OM-GFP), showing delayed FCCP-induced mitochondrial fission in Sept2 depleted cells compared to mock treated cells. White arrowheads point at fission examples, yellow arrowheads to looping. Scale bar: 10 µm, insets are enlarged twofold. (E) Quantification of mitochondrial elongation (long /short axis) in mock treated and FCCP treated U2OS cells showing increased elongation in Sept2 depleted cells compared to mock treated cells, mean of four independent experiments SEM.
Somatic TE insertions in Notch in spontaneous male neoplasia A The fly intestine is maintained by the Intestinal Stem Cells (ISCs).Figure 1A The fly intestine is maintained by the Intestinal Stem Cells (ISCs). In male flies, carrying one X chromosome, the tumor suppressor gene Notch is present in a single copy. Inactivation of Notch in a stem cell (in green) leads to a clonal expansion of the mutant cell and neoplasia. The neoplastic gut region was microdissected together with the head of the same fly. DNA isolated from both tissues was subjected to whole-genome paired-end sequencing. B An Integrative Genomics Viewer (IGV) screenshot of the Notch de novo TE insertion site from sample P47 (clonal neoplasia) and its head control, sample P48. Bars represent sequencing reads. Reads supporting the TE insertion are colored according to homology to a specific TE insertion sequence. Multiple colors at a putative insert site frequently indicate homology to different reference copies of the same TE family. Two types of supporting reads can be seen: soft-clipped reads - spanning the insertion site and mapping partially to the reference genome and partially to the TE, and mate pair support reads - flanking the insertion site and mapping to the reference genome but with mates (not seen) mapping to the TE. C The Notch locus and the identified somatic TE insertion sites indicated with vertical arrows. Black bars represent exons. Insertions in Notch were identified in 3 out of 33 clonal samples from the Pros>2xGFP genetic background and in 1 out of 4 Dl>nslGFP samples. D PCR validation of 4 somatic, neoplasia-specific TE insertions. Primers were designed to target regions flanking the insertion sites. Yellow arrowheads indicate PCR products containing an insertion amplified in the clonal DNA but not in the neighboring gut tissue (non-clonal), head or thorax for the same fly. Short wild-type amplicon was detectable in all samples. Thorax DNA sample was not available for sample P15. E Sanger sequencing of the TE insertion breakpoints in the 3'UTR of Notch from sample P47. The rover LTR element was inserted in a reverse orientation to Notch. The 5' LTR sequence was truncated by 13 bp. Vertical dashed lines indicate insertion breakpoints. LTR - long terminal repeat
Amino acids 400–441 of Smad6 specifically bind to Pellino-1 and inhibit NF-κB signaling.Figure 1A Schematic representation of truncated mutants of the Smad6 MH2 domain and binding to Pellino-1N. HA-tagged Pellino-1N encodes amino acids 1-137 of Pellino-1. B Plasmids encoding truncated mutants of the Smad6 MH2 domain were co-transfected into HEK293 cells with HA-tagged Pellino-1N plasmid. Cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-Myc antibody. Data are representative of at least three independent experiments. IP, immunoprecipitation; IB, immunoblot; TCL, total cell lysates. C, D The SBE-Luc reporter plasmid was co-transfected with an empty vector, Myc-Smad6 (400-441), or full-length Smad6-expressing plasmids into CMT-93 cells, respectively. After 24 h, cells were treated with TGF-β1 for 2 h and luciferase activities were measured and normalized. Data were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.05, ***P < 0.001. C, D The 5xNF-κB-Luc reporter plasmid was co-transfected with an empty vector, Myc-Smad6 (400-441), or full-length Smad6-expressing plasmids into CMT-93 cells, respectively. After 24 h, cells were treated with LPS for 2 h and luciferase activities were measured and normalized. Data were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.05, ***P < 0.001. E CMT-93 cell lines stably expressing Smad6 amino acids 400-441 were treated with LPS for the indicated time and expression of IκBα, IKKα, and phospho-IKKα/β was monitored by immunoblotting. As a positive control, CMT-93 cells were pre-treated with TGF-β1 for 2 h. CMT-93 cells stably expressing the empty vector pMSCV-puro were used as a negative control. β-actin was used as a loading control. All data are representative of three independent experiments.
Figure 5(A HT29 cells were infected with Shigella WT, ∆ospC1, ∆ospD3, ∆ospC1∆ospD3, or ∆ospC1∆ospD3/ospC1 (∆ospC1∆ospD3 complemented with ospC1) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting B) HT29 cells were infected with Shigella WT, ∆ospC1, ∆ospD3, ∆ospC1∆ospD3, or ∆ospC1∆ospD3/ospC1 (∆ospC1∆ospD3 complemented with ospC1) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay (C) HT29 cells were infected with Shigella WT, ∆ospD3, or ∆ospC1. Cell lysates obtained at the indicated time points were subjected to immunoblotting. (D HT29 cells treated with DMSO or caspase-8 inhibitor were infected with Shigella WT, ∆ospC1, ∆ospD3, or ∆ospC1∆ospD3 strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting E) HT29 cells treated with DMSO or caspase-8 inhibitor were infected with Shigella WT, ∆ospC1, ∆ospD3, or ∆ospC1∆ospD3 strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay (F) HT29 cells treated with the control or caspase-8 siRNAs were infected with the indicated Shigella strains and incubated for 8 h. Cell lysates were subjected to immunoblotting. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting.
GRASP55 is required for autophagy‐activated IL‐1β secretion.Figure 4(A) BMM cells were transfected with scramble (Scr) control siRNA or siRNA against GRASP55. After 48 h of transfection, cells were treated with LPS and the day after subjected to 20 μM nigericin in EBSS, and secreted IL‐1β was measured by ELISA. Data represent mean values±s.d. (n⩾3); *P0.05. Inset: Immunoblot analysis of GRASP55 knockdown. (B) Immunofluorescence confocal microscopy analysis of LC3 and GRASP55 distribution. LC3 (green, Alexa488), GRASP55 (red, Alexa568). BMMs were pretreated overnight with 100 ng/ml LPS and either not stimulated (Ctrl) or stimulated (Nig) for 30 min with the inflammasome agonist nigericin (20 μM) in full medium. (C) Line tracings, analysis of fluorescence signal intensity from images in (B). (D) Pearson's coefficients for LC3 and GRASP55 were quantified using SlideBook morphometric analysis software as a measure of adjacency between GRASP55 and LC3 profiles. Pearson's coefficients were derived from three independent experiments with five fields per experiment, for a total of 15 fields contributing to the cumulative result. Figure source data can be found in Supplementary data.
Figure 4Graphical representation of proteinase K assay (i) showing protection of p62/SQSTM1 inside sealed autophagosomes or proteinase K sensitive p62 present within open phagophores. (ii) Graphical representation of sytnatxin17 (STX17) translocation to completed autophagosomes and not phagophores. Autophagosomes are identified as being both LC3B and STX17 positive vesicles. U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS were stimulated with complete medium, (CM), 4 h starvation (EBSS) plus BafilomycinA1 (BafA1, 200nM) treatment. Cells were centrifuged and resuspended in PBS digitonin, spun, washed and the membrane fractions incubated with proteinase K with and without 0.1% TritonX-100. Sampels were then subjected to western blotting using anti-p62/SQSTM1 and anti-HA (ATG2A) antibodies. Percentage p62/SQSTM1 remaining was calculated using densitometry analysis. Blots are representative of n=3 independent experiments. U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS and stably expressing GFP-Syntaxin17 (STX17) were stimulated starvation (EBSS) plus BafilomycinA1 (BafA1, 200nM) for 4 hours to stimulate autophagosome generation and prevent their degradation in the lysosome. Cells were fixed and immune-stained for LC3B (magenta). DAPI was included (blue) to mark the DNA/nucleus. Confocal analysis of LC3B and GFP-STX17 (green) localization was performed. Closed arrows (ATG2A/B DKO and DKO + ATG2A mLIR) highlight aggregate structures. Open arrows (DKO + ATG2A-WT and ATG2A-mYFS) highlight STX17/LC3B positive vesicles. Scale bar 10µm.   Quantification of (C) expressed as a percentage of cells with STX17/LC3B positive vesicles. Each symbol represents a single field of cells with 5-10 cells per field. A total of 300-600 cells were analysed over n=3 independent experiments. Data is shown as mean ± SD.
Figure 3The formation and utilization of lipid droplets in eukaryotes. Five µM combination of DGAT inhibitors (DGAT1 inhibitor A922500 and DGAT2 inhibitor PF-06424439), 40 µM ATGL inhibitor atglistatin (Atg) or 5 µM MAGL inhibitor MJN110 were added to the bone marrow polarization system in the presence of 40 ng/ml GM-CSF and indicated compounds for 7 days. The oxygen consumption rate (OCR) of 1×105 purified Gr1-CD11b+ cells was monitored as described in Figure 2. The basal OCR, basal ECAR (extracellular acidification rate), spare respiratory capacity, proton leak, ATP production and maximal respiration based on the OCR value were quantified. The polarization state was evaluated via the expression of CD206. T cell proliferation assay was performed employing co-culture with purified CD4+ T cell in the variant ratios. The lipid droplets accumulation was determined by BODIPY staining. The percentage of divided cells and proliferation index were calculated. Nitric oxide (NO) concentration in the co-culture supernatant was quantified by Griess reaction.
Antiviral effects of steroids on MERS-CoV infection.Figure 1:(a) Effect of steroids on MERS-CoV replication. Vero cells were infected with MERS-CoV at MOI = 0.01 in the presence of steroids for 24 h. The viral titer in the cell supernatant was quantified by standard plaque assay using Vero/TMPRSS2 cells. Cell viability in the presence of steroids was quantified by WST assay at 24 hours post-infection (hpi). (b) Ciclesonide escape mutant. Vero cells treated with ciclesonide were infected with parental MERS-CoV or ciclesonide escape mutant. Viral titer was measured as described in panel a. (c) Vero cells were infected with the parental MERS-CoV/EMC strain (Re-EMC/MERS) or the recombinant mutant strain (Re-Nsp15-A25V) with an amino acid substitution at A25V in NSP15. Viral titer was measured as described in panel a.
A-BRD is an epigenetic reader of H3K14ac that mediates the BAZ2A association with H3K14ac-chromatin domains in PC3 cells A.Figure 1A. Heatmap profiles of BAZ2A bound regions in PC3 cells and the corresponding signals of input, BAZ2A-BRD mutant (Y1775F), H3K14ac and H3K27ac. Data were ranked to BAZ2A-binding in PC3 cells. B. Validation of ChIPseq results by anti-HA ChIP-qPCR of PC3 wild-type (WT) and a PC3 cell line that expresses endogenous BAZ2A with a FLAG-HA tag (PC3-H/F-BAZ2A). GAPDH promoter represents a region not bound by BAZ2A. Average values of three independent experiments. Data were normalized to input and to AOX1 levels. Statistical significance (P-values) was calculated using two-tailed t-test (*<0.05, **< 0.001, ns: not significant). Error bars represent SD. C. Wiggle tracks displaying BAZ2A-bound regions, H3K14ac, H3K27ac, and input at AOX1, EVX2, and GAPDH. D. Pearson correlation heat map of BAZ2A-bound regions and histone marks. Data for BAZ2A, H3K4me3, H3K27me3, H3K27ac, H3K14ac are from this work. H3K9me3, H3K9me2, H3K4me1 were obtained from ENCODE. E. Images of histone peptide arrays probed with 10nM of recombinant BAZ2A-BRD wild-type (GST-BAZ2A-BRDwt) and mutants (GST-BAZ2A-BRDY1830F or GST-BAZ2A-BRDN1873L). Visualization of binding was performed by incubation with anti-GST antibodies and imaged on Odyssey Infrared Imaging System. Blue circles mark peptides recognized by BAZ2A-BRD (i.e. H3K14ac), whereas black circles show some of the acetylated peptides not recognized by BAZ2A-BRD (H3K27ac and H3K9ac). Right panel shows heatmap of the relative binding intensity of BAZ2A-BRD against modified histone peptides. Binding intensity was calculated as average of fold change from peptides signal over background controls of 2 different arrays. F. BAZ2A read coverage quantification at ±1Kb from peak summit of BAZ2A-bound regions identified in PC3 cells and the corresponding reads for BAZ2A-BRD mutant (Y1775F) in PC3 cells. Statistical significance (P-values) was calculated using two-tailed t-test (***< 0.001). Data information: Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range. G. Regions bound by BAZ2A in PC3 cells are enriched in H3K14ac. Read coverage quantification for H3K14ac levels at regions bound by BAZ2A in PC3 cells (BAZ2Awt and BAZ2A-BRD mutant (Y1775F)) at ±1Kb from BAZ2A peak summits. Statistical significance (P-values) was calculated using two-tailed t-test (***< 0.001). Data information: Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range.
a) Miz1ΔPOZNes mice move slowly and lack motor coordination.f1(a) Miz1ΔPOZNes mice move slowly and lack motor coordination. The panel shows the average speed of movement and time on a rotarod (maximum: 300 s) maintained at constant speed of control and age-matched Miz1ΔPOZNes mice. The average age of mice tested was 16 months. P-values were calculated using an unpaired two-tailed Student's t-test (the number of animals tested is shown below each graph). (b) Defect of cerebellar growth in Miz1ΔPOZNes mice. The panels show sagittal sections of brains of control and Miz1ΔPOZNes mice at the indicated age. Dotted lines show the circumference of the cerebellum. For each mouse, the cerebellar surface area was determined; numbers show the ratio between control and Miz1ΔPOZNes mice. The number of mice analysed for each genotype is indicated on the right. Scale bar, 1 mm. (c,d) Histology of the cerebellum. Sections of the cerebellum of 11-month-old control (c) and Miz1ΔPOZNes (d, please note different scale) mice were stained with antibodies directed against calbindin, a marker for PCs, and glial fibrillary acid protein (GFAP), a marker for Bergman glial cells. Boxes indicate the area of a detail picture in the overviews. GFAP staining is shown for adjacent section (arrowheads: regions of missing PCs). Scale bar, 500 μm (upper panels) and 100 μm (lower panels). (e) Enhanced apoptosis in the granular layer of cerebella in Miz1ΔPOZNes mice. The upper panel shows representative TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) staining of cerebella of age-matched control and Miz1ΔPOZNes mice. The lower panel shows a quantification of staining observed in four control and five Miz1ΔPOZNes mice. Error bars show s.e.m. P-value was calculated using an unpaired two-tailed Student's t-test.
Figure 3Freezing phenotype (A) of Col-0, ost1-3, atann1, and atann1 ost1-3. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min). Representative pictures are shown in (A). survival rate (B) and ion leakage (C) of Col-0, ost1-3, atann1, and atann1 ost1-3. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min). OST1 gene expression D) in Col-0, OST1-Myc, atann1 and OST1-Myc atann1. protein levels of OST1 and AtANN1 (E) in Col-0, OST1-Myc, atann1 and OST1-Myc atann1. OST1 and AtANN1 proteins were detected with anti-Myc and anti-AtANN1 antibodies. HSP90 was used as a control. Freezing phenotype (F) of Col-0, OST1-Myc, atann1 and OST1-Myc atann1. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, -5°C, 30 min; CA, -8°C, 40 min). Representative pictures are shown in (F). survival rate (G), and ion leakage (H) of Col-0, OST1-Myc, atann1 and OST1-Myc atann1. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, -5°C, 30 min; CA, -8°C, 40 min).