EMBO/sd-character-level-ner huggingface dataset:
a) BJAB and Jurkat cells were treated with vehicle or chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by Fas ligand (1.figf3(a) BJAB and Jurkat cells were treated with vehicle or chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by Fas ligand (1.5 ng ml−1). Cell viability was determined by MTS 24 h later (percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 0.0058). (b) Immunoblots of cells in a,c probed for the indicated antibodies. CQ, chloroquine (c) BJAB and Jurkat cells were treated with chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by treatment with Fas ligand (4 ng ml−1) for 24 h (same technical replicate as a). Cells were then re-plated at low density in growth media and allowed to recover for 5 days, and then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0024). (d) BJAB and Jurkat cells were transduced with the indicated lentiviral shRNA constructs, followed by 3 days of puromycin selection. Selected cells were plated and treated with Fas ligand (1.5 ng ml−1) or TRAIL (4 ng ml−1) for 24 h and viability was assessed by MTS (percentage of control (no ligand), mean ± s.e.m., n = 3, *P = 1.7×10−4, **P = 0.024). (e) Immunoblots for Atg5, Atg7 and p62 confirm protein depletion and autophagy inhibition in d,f. (f) BJAB and Jurkat cells expressing control or Atg5 shRNA were treated with Fas ligand (15 ng ml−1) or TRAIL (12.5 ng ml−1) for 24 h (same technical replicate as d). Cells were then re-plated at low density and allowed to recover for 5-6 days, and then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0089). Atg7 knockdown data were not included for long-term viability owing to the growth suppressive effect of Atg7 knockdown in the absence of drug treatment. Uncropped images of blots are shown in Supplementary Fig. 6.
Figure 6Model of ubiquitin-dependent BRCA1 recruitment to DSB sites. BRCA1 localizes at DSB sites via two redundant ubiquitin (ub)-dependent processes. The first mode is mediated by the ANK-BRCT domain of BARD1, also known as BUDR, which recognizes H4K20me0 nucleosomes modified with H2A-K13/15ub, the product of RNF168. Our work is consistent with a role for the E3 ligase activity (green) for promoting this binding event. The second mode on BRCA1 recruitment is dependent on the BRCT domains of BRCA1 binding to BRCA1-A whose RAP80 subunit recognizes Ly63-linked ub chains (ubK63), most likely on histone H1.
a) Magnified view showing the detailed interaction around Leu374(p).f3(a) Magnified view showing the detailed interaction around Leu374(p). (b) Magnified view showing the detailed interaction around Tyr380(p). (c) Magnified view showing the importance of main-chain hydrogen bonds of the Galbi peptide. (a-c) Red dashed lines designate hydrogen bonds. (d) An FP assay using FITC-labelled Galbi peptides, including mutants (L374A, Y380F and Y380A) against an increasing concentration of GAL8. (e) An FP assay using a FITC-labelled Galbi peptide against an increasing concentration of GAL8 mutants (E268A, I270A, Y272A and Trp317). (d,e) The KD values were calculated from at least three independent experiments. ND, not determined.
Figure 3(A) MDA-MB-468 cells were treated for 24h with vehicle, PI3Kβi (AZD8186 250 nM), EGFRi (gefitinib 3 μM) or cetuximab (100 μg/ml), alone or in combination. The cell lysates were probed with the indicated antibodies. (B) MDA-MB-468 cells were treated for 24h with vehicle, AKTi (MK2206 450 nM), gefitinib (3 μM) or cetuximab (100 μg/ml), alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies. (C, D) HCC70 (C) and ZR-75-1 (D) parental cells or PI3Kβi-Res (derivative cells with acquired resistance to AZD8186), or AKTi-Res (acquired resistance models to MK2206) were treated with vehicle, PI3Kβi (AZD8186 250 nM) or AKTi (MK2206 1 μM) for 24 hours. The whole cell lysates were then probed with the indicated antibodies. Spliced images of parental and resistant paired samples were taken from the same original blot and blots showing P-S6 and S6 tot in (D) have been cut and reassembled for figure purposes. (E) HCC70 parental, PI3Kβi-Res or AKTi-Res cells were treated with vehicle, AKTi (MK2206 250 nM) or gefitinib (3 μM), alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies. (F) HCC70 MK res (acquired resistance models to MK2206) were treated with serial dilutions of gefitinib, alone or in combination with MK2206 (810 nM), as indicated, and viability measured after 4 days of treatment. Average ± SD of triplicates and representative of two independent experiments. (G) HCC70 AZD res (acquired resistance models to AZD8186) were treated with serial dilutions of gefitinib, alone or in combination with AZD8186 (270 nM), as indicated, and viability measured after 4 days. Average ± SD of triplicates and representative of two independent experiments. (H,I) p110β co-immunoprecipitates with EGFR in MDA-MB-468 (H) and in HCC70 (I). Cells were pre-treated with different inhibitors, including vehicle, pan-PI3Ki GDC0941 (1 μM for MDA-MB-468 or 0.5 μM for HCC70), EGFRi (gefitinib 3 μM), PI3Kβi (AZD8186 250 nM) or a combination of EGFRi and PI3Kβi. Cell lysates were incubated with IgG control or anti-EGFR antibody and the immuno-complexes or the total lysates were immune-blotted with the indicated antibodies. (J) EGF-induced increase in phospho-AKT is dependent on p110β kinase activity. MDA-MB-468 were starved in 0% FBS and pre-treated with vehicle or PI3Kβi (AZD8186 250 nM) for 1h. Cell lysates were probed with the indicated antibodies. Phospho-AKT and pan-AKT bands were quantified by the use of ImageLite software: the ratio of phospho-AKT to pan-AKT normalised to the control (left hand lane) is shown.
Atg1 induces myosin II activation and spaghetti‐squash activator (Sqa) identification.Figure 1(A-F) Atg1‐induced myosin II activation depends on the kinase activity of Atg1. Third‐instar wing imaginal discs from ptc‐GAL4 UAS‐GFP controls or flies expressing indicated transgenes were stained with phospho‐MRLC (blue) and TRITC‐labelled phalloidin (red). Low level of phospho‐MRLC staining was observed in controls (A) and cells overexpressing kinase‐deficient Atg1‐KR (C), whereas a robust increase in phospho‐MRLC was found in cells overexpressing Atg1 (B). Co‐expression of Atg1 and spaghetti‐squash (A20A21) exhibited low level of phospho‐MRLC staining (D). Atg1‐induced high level of phospho‐MRLC staining was not suppressed by expression of caspase inhibitor p35 (E) or by depletion of Atg12 (Atg12RNAi) (F). Bar, 20 μm. (G) Schematic diagram of Drosophila and mammalian MLCK family. The quantities within the kinase domains indicate the degree of amino acid identity to the kinase domain of Sqa (CG1776). Ank, ankyrin repeats; Death, death domain; Fn, fibronectin domain; Ig, immunoglobulin domain; NLS, nuclear localization signal. (H) Sqa, but not Atg1, directly phosphorylated Sqh in vitro. Flag-Atg1, Flag-Atg1‐KR, HA-Sqa and HA-Sqa‐KA were immunoprecipated from lysate of transfected cells and incubated in an in vitro kinase reaction mixture containing [γ‐32P]ATP and bacterially expressed recombinant wild‐type Sqh or SqhA20A21. As shown on the autoradiogram (top panel), wild‐type but not the kinase‐deficient Atg1 (Atg1‐KR) and Sqa (Sqa‐KA) was autophosphorylated. No phosphorylation was seen with SqhA20A21. The equal input of His‐fusion proteins is shown on the Coomassie staining. Anti‐Flag and anti‐HA immunoblottings (IBs) were used as controls to quantify the amount of proteins precipitated.
Figure 2RPE cells were transfected with control or FAM110A siRNA, whole cell lysates were prepared after 48 h and indicated antibodies were tested in immunoblotting. RPE cells were released for indicated times from a thymidine block and collected cells were analyzed by immunoblotting and FACS. Nocodazole was added after 4 h to arrest cells in mitosis. Alternatively, RO3306 inhibitor was added to prevent mitotic entry. Where indicated sample was collected by mitotic shake-off (MS) to obtain pure mitotic population. Samples were analyzed by immunoblotting (upper panel). Staining for pS10-H3 was used as a marker of mitosis, TFIIH as a loading control. In parallel, cell cycle progression was followed by flow cytometry using DAPI and mpm2 staining. Representative plot is shown, M indicates percentage of mitotic cells (middle panel). Signal of the rabbit FAM110A antibody was quantified in three independent replicates and normalized to the loading and to the thymidine arrested sample (lower panel). Bars show median ± SD. RPE cells were arrested in mitosis by nocodazole, collected by mitotic shake-off and released to fresh media for indicated times to allow mitotic exit. Loss of pS10-H3 signal was used as marker of mitotic exit. RPE cells arrested in G2 by RO3306 (G2) or in mitosis (M) by nocodazole treatment were extracted and endogenous FAM110A was immunoprecipitated using rabbit polyclonal antibody immobilized on pA/G beads. Beads were incubated with mock or with lambda phosphatase for 20 min at 30°C. Proteins were separated by SDS-PAGE and detected by immunoblotting. RPE cells transfected with control or FAM110A siRNA were fixed with 4 % PFA, probed with rabbit polyclonal antibody against FAM110A and mouse monoclonal to γ-tubulin, and were analyzed by confocal microscopy. Shown is maximal projection of representative mitotic (upper panel) and interphase (lower panel) cells. Scale bars indicate 10 μm. Quantification of the signal at spindle poles from (E). Signal intensity of the rabbit polyclonal antibody to FAM110A was determined in regions positive for γ-tubulin. Each dot represents a single spindle pole. Bars show median ±SD. Significance was evaluated by a t-test (n=3), ****p<0.0001. Asynchronously growing RPE cells were fixed and probed with the rabbit polyclonal antibody to FAM110A. Shown are representative images of cells in various phases of mitosis. Individual z-stacks were de-convoluted using Huygens Deconvolution Software, processed sequentially and are presented as maximum projections. Scale bars indicate 10 μm.
Figure 2A) The schematic diagram shows the genomic location and splicing patterns of circFNTA (hsa_circ_0084171). B) The relative expression of circFNTA was elevated in multiple BCa cell lines (T24, J82, UMUC3, and 5637) compared to normal bladder epithelial cell line SVHUC as shown by qRT-PCR. C) The expression of circFNTA and GAPDH mRNA in J82 and UMUC3 cells treated with or without RNase R was detected by qRT-PCR. D) RNA Fluorescence in situ hybridization (FISH) demonstrated that circFNTA was predominantly localized to the cytoplasm of J82 and UMUC3 cells. DAPI was mainly localized to the cytoplasm. Scale bar, 20 μm. E) Two shcircFNTA plasmids using PLKO.1 vector were transfected into UMUC3 cells. The knock down efficacy was measured by qRT-PCR. F) The structure of PWPI-oecircFNTA, PWPI-control-vector and PWPI-oelinearFNTA overexpression constructs. G) The qRT-PCR results show oecircFNTA efficacy in J82 cells compared with PWPI vector (left panel) and oecircFNTA or oelinearFNTA in J82 cells (right panel). Transwell invasion assays showed that shcircFNTA can decrease UMUC3 cell invasion (H) more profoundly than oelinearFNTA as compared with the oevector group. Scale bar, 100 μm. Transwell invasion assays showed that shcircFNTA can increase J82 cells invasion (I) more profoundly than oelinearFNTA as compared with the oevector group. Scale bar, 100 μm. J) MTT assays showed that oecircFNTA enhances J82 cells chemo-resistance to cisplatin (0, 1, 2, 5, 10 μg/ml were tested). This effect was more obvious in the oecircFNTA group as in the oelinearFNTA group (upper panel). MTT assays showed that shcircFNTA increases UMUC3 cells chemo-sensitivity to cisplatin (lower panel). K) FISH analyses revealed that circFNTA was significantly upregulated in BCa tissues as compared with the adjacent para-tumor tissues (N=41 patients) and representative images are shown. Scale bar, 250 μm. L) The qRT-PCR assays also demonstrated that circFNTA is significantly elevated in fresh BCa tissues compared to para-tumor tissues (N=23 patients).
Figure 6(A, B) mCherry-LC3 and MTS-TagBFP were expressed in MTPAP KD and control cells. Cells were incubated with valinomycin for the indicated times and immunostained with anti-NDP52 antibody. Images are representative of two independent experiments. Yellow arrows indicate co-localization of MTS-TagBFP, mCherry-LC3 and NDP52. White arrows indicate co-localization of MTS-TagBFP and NDP52 (not mCherry-LC3). Scale bars, 10 µm. Co-localization of mCherry-LC3 and NDP52 on mitochondria is quantified in (B). At least ten cells from two independent experiments were quantified. Values are the means ± SEM. **p < 0.01 vs control, determined with one-way ANOVA followed by the Student"s t MTPAP KD and control cells expressed mCherry-NDP52 or empty vector (C Lysates from cells treated with 1 µM valinomycin and 100 nM bafilomycin A1 for 2 h were subjected to immunoprecipitation using anti-RFP beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments MTPAP KD and control cells expresse mCherry-LC3 (D). Lysates from cells treated with 1 µM valinomycin and 100 nM bafilomycin A1 for 2 h were subjected to immunoprecipitation using anti-RFP beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments (E) MTPAP KD and control cells were incubated with valinomycin and bafilomycin A1 for 2 h. Cell lysates were subjected to immunoprecipitation using an anti-NDP52 antibody or control IgG. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments (F) GST-NDP52 conjugated with glutathione-Sepharose resin was incubated with purified recombinant LC3 and MTPAP∆MTS∆C. The pulled down complex was subjected to western blotting. Data are representative of three independent experiments
miR-132 promotes protective immunity to L.Figure 5Liver LDU (Leishman Donovan units) at day 28 in infected WT mice treated with anti-IL-10R antibody or isotype control antibody (left panel, n=5 mice per group), or at day 21 and day 28 from WT (blue), IL-10+/- (open green circles) and IL-10-/- (filled green circles) mice (right panel n= 3-6 mice per group) Data information: Significance determined by unpaired t-test * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Day 28 splenic parasite burdens expressed as LDU with each data point representing an individual mouse in WT (blue) and miR-132-/- (miR-132-/-; red) mice. Data from 4 independent infection experiments.   Mean WT and miR-132-/- spleen parasite burdens from the 4 independent experiments shown in (B). Lines link individual experiments.   Splenic parasite burdens relative to WT group (WT mean = 1) for each of the 4 experiments shown in (B), with each data point representing individual mouse.   Data information: Significance determined by unpaired t-test, and in (C) by paired t-test of mean values. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Spleen size expressed as % body weight for d0 (naïve) or day 28 L. donovani infected WT (blue) and miR-132-/- (miR-132-/-; red) mice.   Liver size expressed as % body weight for d0 (naïve) or day 28 L. donovani infected WT (blue) and miR-132-/- (red) mice.   Data information: Significance determined by unpaired t-test * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Figure 5A. CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs were FACS-purified from total epidermis (d0) to ≥98% purity post sorting (d0 post-sort), and grown 14 days in 3C cultures (d14 3C). A significant population of CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs is generated from both purified populations after 14 days. A representative of 3 independent experiments is shown (mean ±SD). B-C. Time course analyses of 3C cultures established from FACS-purified populations of CD34+α6+ HFSCs (B) or CD34¯α6+ non-HFSCs (C) from 3C cultures. Both cultures reach population equilibrium between day 6 and 10 (n=4; mean ±SD). D-E. Simulated population proportions of CD34+α6+ HFSCs (D) and CD34¯α6+ non-HFSCs (E) from the mathematical model describing behavior of pure cell populations. The model parameters were estimated from the experimental data in B-C (shown in grey as reference). F. Schematic illustration of lineage tracing experiments. G. Lineage tracing of FACS-purified CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs from EGFP¯ and EGFP+ 3C cultures, respectively. CD34+α6+ and CD34¯α6+ were mixed at the indicated ratios at d0 and EGFP expression was used to trace the cells of origin over time by flow cytometry. Presence of CD34+α6+ EGFP+ cells in cultures at d10 indicates non-HFSC to HFSC interconversion. H. Lineage tracing as in (F) shows that 3C cultures reach equilibrium between day 6 and 10 independent of the initial proportions of HFSCs and non-HFSCs. I. Quantification of the proportion of CD34+α6+ cells that maintain their fate over time (experiments with both EGFP+ or EGFP¯ labeled cells are indicated). J. Quantification of the proportion of CD34¯α6+ cells that maintain their fate over time (experiments with both EGFP+ or EGFP¯ labeled cells are indicated). Data in panels G-J show n=4; mean ±SD. See also Appendix figure S5.
Timeline of events.Figure 1(A) Series of events in MPP-R and MPP-D (boxes with solid and dashed outline, respectively) preceding the outbreak in MPP-R. The encounter between MPP-R and MPP-D workers which may have initiated the outbreak in MPP-R is shown in the grey box to the right. * quarantine in MPP-D involved all positive cases(n=94) as well as those among the 185 workers with negative test results who had been directly employed in meat processing. Negatively tested employees with other roles (e.g., administrative or security staff) were not quarantined. (B) Events in MPP-R during the outbreak in late May. The three consecutive days during which the index case B1 worked in the early shift and thus the time period during which work-related exposure may have occurred is highlighted in blue. (C) Events in MPP-R during June. Risk- and evidence-based sampling during early June was performed by health authorities, general practitioners, as well as the internal test center from MPP-R. While we do not have exact information regarding the total number of positive cases for this time period, we have indicated a minimum incidence number above the timeline (110) based on publicly available reports from the local health authorities (https://www.kreis-guetersloh.de/aktuelles/corona/pressemitteilungen-coronavirus/). Boxes below the timeline mark positive cases from internal MPP-R testing that were subjected to viral genotyping. Cases designated as pork processing workers were directly associated with meat processing (deboning, shearing, packaging), while internal employees denotes individuals working in areas such as the convenience food section, technical operation, or occupational safety.
Figure 6Gene ontology analysis of genes downregulated in slow/slow neurons showing the top ranked cellular component (CC), biological processes (BP) or molecular functions (MF) GO categories. Box plot showing the length of upregulated (UP), downregulated (DOWN), not affected genes (ALL), and pre-mRNAs affected by alternative splicing (AE, for Alternative exons) for ESCs (left panel); length of upregulated (UP), downregulated (DOWN), not affected genes (ALL), pre-mRNAs affected by alternative splicing (AE, for Alternative exons), and not affected genes that are only expressed in neurons (but not in ESCs) (ALL_notESC), for Neurons (right panel). n.s., p-value>0.05, **p<0.005; ****p<0.0001 as determined by Mann-Whitney t-test. Boxes delimit the first and third quartiles. The horizontal lines represent the data medians. Whiskers are drawn down to the 1st and 99th percentile. Percentage of genes that are downregulated in ESCs and neurons; plotted as a sliding window of 100 genes by length.
Model showing how kinetochore-microtubule attachments contribute to acentrosomal spindle bipolarization(A) K-fibers are connected to MTOCs and extend to spindle poles.Figure 5(A) K-fibers are connected to MTOCs and extend to spindle poles. Oocytes 6 hours after NEBD were fixed following cold treatment. Microtubules (α-tubulin, green), MTOCs (pericentrin, magenta), kinetochores (ACA, red) and DNA (Hoechst333342) are shown. Five oocytes from 2 independent experiments were analyzed. Scale bar, 10 μm. (B) A majority of MTOCs are attached to K-fibers. The volumes of individual MTOCs were measured after 3D reconstruction. The sum volume of MTOCs attached to K-fibers relative to the total volume of MTOCs was calculated for each oocyte (n=5 oocytes from 2 independent experiments). (C) K-fibers extend to spindle poles. The length of K-fibers and distance between kinetochores and a spindle pole (KT-to-Pole distance) were measured. K-fiber length relative to the KT-to-Pole distance was calculated (n=90 K-fibers of 5 oocytes from 2 independent experiments). (D) Model summarizing the results of this study. In early phases of acentrosomal spindle assembly, spindle bipolarizers, including Kif11, HURP, NuMA, HSET, and the kinetochore Ndc80 complex that recruits Prc1, establish spindle bipolarity by promoting MTOC sorting and spindle elongation. Meanwhile, the stability of kinetochore-microtubule attachments gradually increases, depending on the dephosphorylation of kinetochore components including Ndc80. As spindle bipolarity is established, stable kinetochore-microtubule attachments spatially confine MTOCs at spindle poles and limit spindle elongation.
PLT3 PrD domains influence its subnuclear localisation.Figure 6A, Schematic representation of PLT3 protein domains. The areas in red are predicted prion-like domains (PrDs) and were deleted in PLT3ΔPrD-mV. The areas highlighted in yellow contain polyQ-stretches and were deleted in PLT3ΔQ-mV. Representative images of PLT3-mV (B), PLT3ΔQ-mV (C) and PLT3ΔPrD-mV (D) in transiently expressing N. benthamiana leaf epidermal cells. E, F, PLT3-mV (E) and PLT3ΔPrD-mV (F) expression driven by the PLT3 endogenous promoter in lateral root primordia of plt2, plt3 double mutant Arabidopsis roots. G-I'', Co-expression of PLT3-mV (G), PLTΔQ-mV (H) and PLT3ΔPrD-mV (I) with WOX5-mCh (G', H', I') in transiently expressing N. benthamiana leaf epidermal cells. J-J''', Expression of PLT3-mV (J) in transiently expressing N. benthamiana leaf epidermal cells in combination with RNA staining with EU (18 h), visualised by click-reaction with Alexa Fluor® 555 (J') and a DNA staining with DAPI (J''). mV = mVenus; PrD = prion-like domain; AP2 = APETALA2 domain; NLS = nuclear localisation signal; EU = 5-ethynyl-2'-uridine. Scale bars in (B-J''') represent 5 µm.