EMBO/sd-character-level-ner huggingface dataset:
Workplace location and infection events in the beef processing plant.Figure 3(A) Distance (in meters) of PCR-tested workers from the suspected index case B1 at the workplace. For workers without fixed position in the beef processing plant (marked with an asterisk) coordinates indicate estimated average location during the early shift. Squares and diamonds denote prototype or variant SARS-CoV-2 genotypes, respectively. Filled blue circles denote cases for which viral genomes were not sequenced (i.e., workers tested positive after 5/25/2020). (B) Top panel: Observed accumulated percentage of positive cases (red line) within the indicated distance from the suspected index case. The gray dashed line shows the average infection rate that would be expected for a random spatial distribution of positive cases. Bottom panel: -log10 p-value for the frequency of accumulated positive cases within the given distance being significantly higher than expected based on a random spatial distribution of positive cases Only employees with fixed work positions were included in the calculation. (C) Values on the x-axis show infection rates among members of shared apartments, bedrooms or carpools. Values on the y-axis reflect -log10 p-values for the hypothesis that the infection rate within a given unit is higher than expected based on a random distribution of positive cases among all workers sharing one or more unit Infection rates and p-values associated with the 8 m work area around the index case are shown for comparison. Bubble sizes indicate the total number of individuals within each unit or area. All data points with significant p-values (<=0.05) are labeled with unit or area id, positive and total number of associated individuals, and p-value.
Reduced levels of separase increases susceptibility to oncogenic transformation in mice.Figure 8(A) Transformation susceptibility of primary MEFs. SEPARASE+/+ versus SEPARASE+/- MEFs were infected to express E1A and hRasV12 and then subjected to a colony formation assay. Shown are representative images (left) and the graphical representation of the number of transformed foci (right) from two independent experiments (bars = standard deviations)  (B) Two-stage DMBA (initiation) plus TPA (promotion) skin carcinogenesis. The number and size of skin papillomas are plotted against time for each genotype: SEPARASE+/+ (n=8) versus SEPARASE+/- (n= 11)  (C) Representative hematoxylin- and eosin-stained sections from SEPARASE+/- biopsies. (i) glandular hyperplasia; (ii) benign papilloma; (iii) squamous cell carcinoma. Scale bars are 200 µm in (i) and 500 µm in (ii) and (iii). (D) Quantitative histological assessment of the DMBA-TPA skin cancerogenesis assay. Given are numbers of total, and corresponding percentage in brackets
Lam6 Is Localized to Several Cellular Contact Sites.Figure 2(A) Fluorescence microscopy demonstrates that Lam6-GFP co-localizes with ERMES (Mdm34-Cherry) (yellow arrows) and also localizes to non-ERMES locations in the cell (red arrows). These additional locations co-localized with the NVJ (Nvj1-GFP) as well as with the vCLAMP (GFP-Vps39). Scale bar represents 5 μm. (B) Overexpression and tagging of Lam6 confirmed that it co-localizes with ERMES (Mdm34-GFP) (yellow arrows) as well as to additional contact sites (red arrows), NVJ (marked by Nvj1) and vCLAMP (marked by Vps39). Scale bar represents 5 μm.
Figure 3(A) Nuclear localised Pbl-GFP becomes cytoplasmic in mitotic cells, co-incident with Rok-GFP relocalisation to the plasma membrane. Scale bar ~1µm. n > 5 independent biological replicates. (B) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; purple) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild-type wing epithelial cells, but not those expressing UAS.pbl-RNAi. Phospho-Histone H3 (pH3) staining (green) marks mitotic cell chromosomes. Quantification shown right (n > 10 independent samples per genotype; mean +/- 1 S.D. shown). Scale bar ~1µm. (C) A stripe of expression of ptc.Gal4 UAS.pbl-RNAi in the wing disc leads to both enlarged cells and many basally extruded cells that undergo apoptosis, marked by pyknotic nuclei and loss of aPKC/Dlg staining. Scale bars ~20µm. n > 6 independent biological replicates.
Figure 5A Dual-luciferase assay of TRAF2-, TAB2-, TAB3-, TAK1-, or TAB1-induced NF-κB activation in the absence or presence of Mtb PknG in HEK293T cells. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05. Data were analyzed according to a two-tailed unpaired t-test using GraphPad Prism 7. B IP of TRAF2 and TAK1 by Mtb PknG in U937 cells. Cells were infected with the indicated Mtb strains at an MOI of 1. Non-infected cells were used as controls. After 4 hours, the cells were lysed and immunoprecipitated with the antibody against PknG. The immunoprecipitated proteins were immunoblotted with TRAF2, TAK1, TAB2 (negative control), or TAB3 (negative control) antibodies. C IP of TRAF2 and TAK1 by GFP-tagged Mtb PknG or its truncated forms in transfected HEK293T cells. D Immunoblotting analysis of TRAF2, TAK1, and PknG from lysates of U937 cells infected with the indicated Mtb strains at an MOI of 1 for 0-24 hours. GAPDH was used as the loading control. Densitometric quantification of immunoblots is indicated below the immunoblots. E Immunoblotting of TRAF2, TAK1, and PknG from lysates of U937 cells infected with the indicated Mtb strains at an MOI of 1 for 8 hours. Cells were treated with CHX (50 μg/mL) with or without MG132 (5 μM). Cell lysates were analyzed by immunoblotting for TRAF2 (anti-TRAF2), TAK1 (anti-TAK1), and PknG (anti-PknG). GAPDH was used as the loading control. Densitometric quantification of immunoblots is indicated below the immunoblots.
Figure 3A Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was analyzed by SDS-PAGE and silver staining. The input sample and mock precipitation using total mouse IgG are also shown. The gel slices indicated by boxes were excised and subjected to LC-MS/MS analysis. MS/MS spectrum of the histone H3 peptide corresponding to residues 9-17. The observed y and b ions and fragment map are shown. C Summary of the ubiquitylated peptides identified by LC-MS/MS. D Relative abundance of ubiquitylated lysine residues in chromatin-associated histone H3 fractions. Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was subjected to LC-MS/MS analysis, and the abundance of ubiquitylated lysine residues in each fraction is shown Since short peptides cleaved at unmodified lysine residues could not be efficiently detected in the LC-MS/MS analysis, these data do not accurately reflect the abundance including unmodified lysines.
Bio-ID reveals a network of ATG9A interactors, including multiple trafficking regulators and members of the ULK1 complex.Figure 1(A) HEK-293T ATG9A WT, ATG9A KO or ATG9A KO cells reconstituted with overexpressed HA-ATG9A and HA-ATG9A BirA* were grown in full DMEM media. Endogenous p62/SQSTM1 level was measured by immunoblot (top). Graph below shows quantification of p62 infrared signal normalized to Actin. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA followed by Fisher's LSD tests. ns=p>0.05, *=p≤0.05, **= p≤0.01, ***=p≤0.001, ****= p≤0.0001. (B) HEK-293T parental cells or HEK-293T cells stably expressing HA-BirA* or HA-ATG9A BirA* (stably integrated with lentivirus) were grown in full DMEM media, treated with 50 µM biotin for 12 hrs, followed by detergent lysis and incubation with streptavidin resin. Streptavidin pull-down samples were resolved in a 4-15% gradient gel and coomassie stained. (C) An experimental schematic of Bio-ID workflow. (D) Quantitative proteomics data from three independent experiments were analyzed by volcano plot. Significant interactors were selected based on a cut-off of p≤0.05 (two-tailed heteroscedastic t-test) and ≥2-fold increase in interaction comparing the AUC signal for each peptide from HA-ATG9A BirA* vs HA-BirA* samples. A log 2-fold change =1 and -log p value 0.05 = 1.3 were marked by dash lines on the volcano plot. Significant interactors were colored in salmon and interactors of particular interest were color coded (E) A subset of interactors were validated by immunoblotting with indicated antibodies after the streptavidin pulldown step in panel C. (F) The subcellular localization of significantly scored interactors from three independent proteomics experiments was assigned by using panther GO enrichment analysis (G) A schematic representation of proteins identified by proteomics analysis grouped into protein complexes and associated trafficking pathways. Protein complexes were assembled from the GO enrichment and analyzed using STRING
Proteomics reveal a role for ECE2 in ECM production and integrity.Figure 6Volcano plot visualizes the proteomic analysis from 60d old CO lysates showing proteins with significantly lower and higher expression upon ECE2 KO (n=3 batches of 60d old CTRL and ECE2 KO COs; FDR 0.05; s0 1; dashed lines indicating the cut-off; names of example proteins are shown). Pie chart with proteins downregulated in ECE2 KO COs sorted by relevant categories (Number in the elements = number of proteins falling into each category, with numerous proteins falling into several ones) [50-52]. Volcano plot of proteomic analysis of cell culture supernatant from 55d old CTRL and ECE2 KO COs shows two significantly downregulated proteins (red) in the secretome (n=3 batches of CTRL and ECE2 KO COs; FDR 0.05; s0 1; dashed lines indicating the cut-off). Non-significantly changed proteins of interest are highlighted in green below the cut-off curves. GO cellular component analysis of proteins with dysregulated phosphorylation sites (either found excusively in CTRL or KO or up- or downregulated in ECE2 KO COs relative to CTRL) identified in whole CO proteomic analysis. Schemes summarizing identified phenotypes in in vivo and in vitro model systems upon acute ECE2 KD (E) or chronic inhibition or KO (F) or upon acute ECE2 OX (G) and thus the molecular action of ECE2 (Abbreviations: d = day, pe = post electroporation, VZ = ventricular zone, SVZ = subventricular zone, IZ = intermediate zone, CP = cortical plate, aRG = apical radial glia, bRG = basal radial glia, IP = intermediate progenitor, ECM = extracellular matrix).
Figure 2A Molecular structures of geranylgeranyl pyrophosphate (GGPP) and its biotinylated analogue biotin-geranyl pyrophosphate (BGPP). Geranylgeranyl moiety consists of four repeats of 5-carbon isoprene unit. B Purification strategy for GGTase-III substrate proteins using statin and BGPP. "X" represents a putative substrate protein of GGTase-III. HMG-CoA, hydroxymethylglutaryl-CoA. C Identification of Ykt6 as a protein biotin-geranylated by GGTase-III. Cytosolic proteins extracted from statin-treated HeLa S3 cells were applied to GST or GST-GGTase-III affinity columns. Bound proteins were eluted and incubated with recombinant GGTase-III and BGPP. Reaction products were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with horseradish peroxidase (HRP)-labeled avidin to detect biotinylated proteins. Mass spectrometry identified the 25 kDa protein as Ykt6. D Biotin-geranylation of recombinant Ykt6 by GGTase-III. Bacterially produced recombinant untagged Ykt6 (left) was incubated with buffer, GGTase-III, or RabGGTase in the absence or presence of BGPP for 30 min at 37ºC. Biotin-geranylated Ykt6 was detected
Figure 2MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. The reconstitution efficiency (frequency of human among all human and mouse CD45+ leukocytes) is shown in A MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. the average human leukocyte composition in the bone marrow of the mice is shown in B. MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. Tumor volumes over time are presented in C MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. tumor weights and volumes at the study endpoint in D, where each data point represents one tumor. MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. The reconstitution level directly correlates with the tumor weight at the endpoint (E). MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age. The lymphoma burden at the study endpoint was quantified in the spleen (F) and bone marrow (G) by staining for hCD45 (as well as hCD19 and hCD20 for U-2932 and RC-K8, respectively) MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with RC-K8 cells at six weeks of age. The lymphoma burden at the study endpoint was quantified by ZsGreen expression and in the case of RC-K8 cells, also over time by IVIS (H). MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age. The frequency of tissue involvement was assessed by ex vivo IVIS and macroscopic evaluation (I), MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age. the occurrence of clinical symptoms (weight loss, hind leg paralysis) was recorded for each mouse (J).
Figure 4Figure 4. Decrease of nucleoid number without mtDNA depletion in HeLa cells overexpressing the CHCHD10S59L and CHCHD10P34S alleles. A. Transfections were performed with empty vector (EV) or vectors encoding wild-type CHCHD10-FLAG (WT), CHCHD10-FLAG (S59L) or CHCHD10-FLAG (P34S) mutants. Mitochondria were stained with Mitotracker. Cells overexpressing wild-type and mutant CHCHD10 were labeled with FLAG antibodies. Nucleoids were visualized with an antiserum against DNA. Image analysis was performed by confocal microscopy. Scale bar: 10 µm. B-C. The average number (B) and size (C) of nucleoids, labeled with antibodies against DNA shown in A, were quantified for 35 randomly-selected individual cells per each studied cell line from 2 independent experiments. Differences between the cell lines were analyzed by Student's t-test (two-sided): very significant (**: p=0.0280) or extremely significant (***: p=0.0001). D. Mitochondrial DNA content was quantified by real-time PCR. Data were expressed as ratio between mtDNA and nDNA concentration. Results represent the mean of relative PCR ± SD of 3 independent experiments. Statistical analysis were performed by Student's t-test (two-sided).
| NSP5 drives of liquid-liquid phase separation required for viroplasm formation.Figure 5(a) DeePhase analysis of the phase-separating properties of NSP5 (regions with a score of > 0.5, i.e., above the dotted line, indicate residues with high LLPS potential). The CTR region (AA 179-197) is highlighted in red. Note that the amino acid numbering represents an average score with a 30-residue sliding window, and not individual residues. Green boxes denote NSP5 residues of previously shown to be essential for viroplasm-like structure formation when co-expressed with NSP2(Eichwald et al, 2004). (b) EMBOSS protein charge plot shown as an alternating blue (negatively charged) and red (positively charged) regions with a 30-residue sliding window. (c) flDPnn Disorder Propensity Plot of NSP5. Regions with a score above the dotted line are predicted to be disordered. (d) NSP5 model predicted using Alphafold2. Region 179-197 of the CTR within the predicted C-terminal alpha helix is highlighted in red. Top panel, left to right: NSP5-∆CTR labelled with Atto-647 (25 μM) incubated with unlabeled NSP2 (10 μΜ); NSP5-∆CTR in the presence of 10% v/v PEG-20K; and with 5 μΜ of poly-arginine (polyArg). Bottom panel: Atto-647-labelled NSP5-∆CTR (5 μΜ) incubated with unlabeled NSP5 (25 μΜ) and Atto-488 labelled NSP2 (10 μΜ). NSP5/NSP2 droplets containing labelled NSP2 (green) also contain NSP5-∆CTR-Atto647 (magenta), shown along with an image of both 488/647 channels overlaid. Scale bar, 10 µm.
The N-terminal region of TFEB is necessary for interaction with Rag heterodimers and lysosomal localization.Figure 6.(A) Summary of the nuclear and lysosomal distribution of several TFEB amino acid and deletion mutants in ARPE-19 cells treated with either DMSO or Torin-1. (B) ARPE-19 cells were nucleofected with the indicated Rag- and TFEB-expressing plasmids. After 12 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting with antibodies against FLAG and GST (used to detect TFEB and Rag proteins, respectively). (C) FLAG-tagged TFEB-WT or TFEB deletion mutants were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting with antibodies against FLAG, 14-3-3 binding motif, or 14-3-3. (D) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB-WT and TFEB-S3A/R4A mutant upon incubation with DMSO (vehicle) or 250 nM Torin-1 for 1 h. Cells were fixed, permeabilized with 0.2% Triton X-100, and stained with antibodies against FLAG (used to detect TFEB). (E) ARPE-19 cells expressing either TFEB-S211A or TFEB-S211A-Δ30 were double stained with antibodies against TFEB and Lamp1. Regions within the dotted boxes are magnified in the insets.
a,b) dUTX directly interacts with ecdysone receptor (EcR/Usp).f2(a,b) dUTX directly interacts with ecdysone receptor (EcR/Usp). (a) GST and GST-dUTX-NR containing the nuclear hormone domain were used in pull-down experiments with 35S-labelled EcR and Usp in the presence of ethanol control (−) or 10 μM ecdysone (+). The GST input proteins are shown in Coomassie Brilliant blue-stained gel, GST is labelled with arrow head and dUTX-NR with an asterisk. The EcR and Usp inputs are shown labelled with arrows. (a,b) dUTX directly interacts with ecdysone receptor (EcR/Usp). (b) Immunoprecipitation (IP) of EcR and dUTX in SL2 cells. Cells were transfected with HA-dUTX and the cell lysates were immunoprecipitated using anti-HA or a control IgG isotype antibody. The immunoprecipitates were analysed by western blotting using the anti-EcR and anti-HA antibodies. (c) Colocalization of dUTX and EcR on salivary gland polytene chromosomes. Immunostaining of salivary glands polytene chromosome prepared from third instar larvae expressing FLAG-dUTX with dUTX (green) and EcR (red), with Hoechst-stained DNA (blue). Arrows indicate examples of colocalized bands. No signal was detected when immunostaining with secondary antibody only. Scale bar represent 5 μm.
Figure 6Analysis of p62/SQSTM1 levels in patient and control fibroblasts transduced with control or VPS16-expressing lentivirus under basal conditions and after 2 hours of serum-starvation. Summary quantification of (A), normalized to actin (ACTB) and expressed as % of controls (n=3 biological replicates). Analysis of LC3 isoforms in serum-starved fibroblasts transduced with control or VPS16-expressing lentivirus and treated with Bafilomycin A1 (Baf A1), as indicated. Quantification of LC3-II, normalized to levels of LC3-I, actin and expressed as % of controls (n=6 biological replicates). Autophagic flux, calculated as the relative increase in LC3-II ratios upon addition of Baf A1 (data from D; n=6 biological replicates). Analysis of autophagosome numbers in fibroblasts transfected to express mRFP-GFP-LC3. (F) Representative confocal micrographs (optical sections) of fibroblasts imaged live under basal or serum-starved conditions. Scale bar, 20 μm. Analysis of autophagosome numbers in fibroblasts transfected to express mRFP-GFP-LC3. Quantification of autophagosomes, defined as green + red puncta (G) and autophago-lysosomes, calculated as the number of red (but not green) puncta divided by the total number of puncta. Colored horizontal bars denote median values and whiskers 5 and 95 percentiles (n=50-113 cells from a total of 3 independent experiments; for exact values see Appendix Table S1). Analysis of autophagosome numbers in fibroblasts transfected to express mRFP-GFP-LC3. Quantification of autophagosomes, defined as green + red puncta (G) and autophago-lysosomes, calculated as the number of red (but not green) puncta divided by the total number of puncta. Colored horizontal bars denote median values and whiskers 5 and 95 percentiles (n=50-113 cells from a total of 3 independent experiments; for exact values see Appendix Table S1). Analysis of autophagosome numbers in fibroblasts transfected to express mRFP-GFP-LC3. Cell of patient A transfected with mRFP-GFP-LC3, labeled with LysoTracker, and imaged live by confocal microscopy under serum-starved conditions. Insert shows red+blue autolysosomes (arrowheads) and a green+red autophagosome devoid of LysoTracker signal (arrow). Scale bars 10 μm or 2 μm (inserts).
Figure 5(A) Schema of experimental designs and the targets of inhibitors. Serial treatments with CTPI-2 (50 mg/kg/shot/day, i.p.), BMS303141 (100 mg/kg/shot/day, p.o.), A-485 (20 mg/kg/shot/day, i.p.), or TV-3664 (10 mg/kg/shot/day, p.o.) were performed 3, 4, and 5 days after the administration of 5-FU. (B-C) The effects of CTPI-2 (B) or BMS303141 (BMS) (C) on H3K27ac in HSCs in the early phase after the administration of 5-FU. (D-E) The effects of CTPI-2 (D), BMS303141 (BMS) (E), A-485 (D), and TV-3664 (E) on HSC expansion in the early phase after the administration of 5-FU. Numbers in the dot plots represent the frequencies of the indicated fractions within total BM cells. (F and G) Average plot (top) and heatmap (bottom) of H3K27ac reads in the cis-regulatory regions of genes belonging to the gene set "HALLMARK_MITOTIC_SPINDLE" (F) or "HALLMARK_G2M_CHECKPOINT" (G) in HSCs in the steady state or early phase. (H) The enrichment of the gene set "GOBP_FATTY_ACID_BIOSYNTHETIC_PROCESS" within differentially expressed genes of the steady state vs the early phase. (I) Average plot (top) and heatmap (bottom) of H3K27ac reads in the cis-regulatory regions of genes having the GO term "fatty acid biosynthetic process" in HSCs in the steady state or early phase.
Mutation of the human IFITM3 endocytosis motif converts IFITM3 into an enhancer of SARS-CoV-2 Spike-mediated cell-to-cell membrane fusion.Figure 6U2OS cells transfected with EGFP and the indicated IFITM3 constructs or vector control were mixed Calu-3 cells transfected with mCherry and SARS-CoV-2 Spike (S) or vector control. Cells were co-cultured for 24 h and imaged by fluorescent microscopy to visualize EGFP and mCherry as well as nuclei via Hoechst dye. A) Representative example fluorescent imaging of the indicated co-cultures showing the presence of EGFP/mCherry double positive cell syncytia dependent upon S expression as well as varying effects of the IFITM3 constructs. Scale bar indicates 50 μm. B) Quantification of nuclei per EGFP/mCherry double positive cell syncytia in co-cultures involving the indicated IFITM3 constructs as compared to vector control. Bars depict averages of three independent experiments with circles representing the individual data points. For each condition, nuclei within 50-75 individual syncytia were counted. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.