EMBO/sd-character-level-ner huggingface dataset:
Figure 2(A) Shown are levels of two EGF-regulated lncRNAs: the EGF-downregulated lncRNA called LIMT (left) and the up-regulated lncRNA called LOC388796 (right). RNA abundance was determined using microarrays (red) and real-time qPCR (blue) and presented as fold-change relative to time zero. Beta-2-microglobulin was used for normalization. (B) Shown are Kaplan-Meier plots of overall survival and relapse-free survival for the EGF-regulated lncRNA called LIMT. To obtain the data, we overlapped the list of EGF-regulated lncRNAs with the METABRIC clinical dataset (Illumina platform; left panel) and the KM-plotter dataset (Affymetrix platform; right panel). The red and blue lines of the left panel correspond to high and low expressors, respectively (each shows one third of the population; 1129 out of a total of 1,693 patients). The same applies to the right panel, except that the population was divided into two equal size groups (N=1,660 patients). (C) Coding potentials of clinically significant lncRNAs were calculated using CPAT, and they are individually presented along with the mean coding probabilities of control groups of protein coding RNAs and lncRNAs (in blue). Note that two variants of LINC00472 and LINC00652 are presented. (D) Evolutionary conservation of the primary nucleotide sequences of the clinically significant EGF-regulated lncRNAs was calculated using PhyloP across 100 vertebrates. Individual conservation scores are presented along with that of control groups of protein coding RNAs and non-coding RNAs (in blue).
Figure 1Schematic of flow cytometry sorting of RFP+ and GFP+ cells from asynchronously growing RPE-FUCCI cells. DAPI was used to probe for DNA content. Representative image of FUCCI cells imaged by immunofluorescence microscopy is shown on the left. Scale bar indicates 10 μm. RNA was isolated from asynchronic RPE-FUCCI cells, RFP+ (G1 phase) and GFP+ (G2 phase) cells and analyzed using Illumina HumanHT-12 v4 Expression BeadChip (n=4). Shown are 36 transcripts with fold change expression > 8 between G2 and G1 cells and q<0.05. Color scale of the row Z score is shown on the left. RFP+ (G1 phase), GFP+ (G2 phase), and GFP+/RFP+ (S phase) RPE-FUCCI cells were sorted by flow cytometry, RNA was extracted and analyzed using qRT-PCR (n=3). Expression is normalized to GAPDH mRNA. Bars show mean ± SD.
Wound-induced tumour formation comprises a fibrotic response.Figure 2a. Immunofluorescent images of skin sections from PDGFRα-eGFP reporter mice with normal (WT, n=4), inflamed (InvEE, n=4), wounded (d14 post-wounding; wound, n=4), or tumour-associated (Papilloma, Pap d17 and d30 post-wounding, n=4 per condition) skin. Nuclei were stained with Dapi (4', 6-diamidino- 2- phenylindole; blue). Dotted line represents epidermal-dermal boundary. Scale bars: 50 μm. b. Quantification of the number of fibroblasts present in different skin conditions (n=4 mice per condition; ** p<0.01; *** p<0.001; **** p<0.0001; One-way ANOVA testing). Data represent means of multiple microscopic fields ± S.E.M. c. Herovici staining of sections of normal (WT), inflamed (InvEE), wound, or tumour-associated (Papilloma) skin. Scale bars: 100 μm. d. pNA (p-nitroaniline) release assessed by spectrophotometric analysis as a measure of serine protease activity in normal (WT, n=5), inflamed (InvEE, n=5) and tumour-associated (Pap, n=7) skin. (* p<0.05; One-way ANOVA testing). Data represent means ± S.E.M.
Figure 3miRoot snapshot of cell-type specific miRNAs showing, in this case, a sum group of the miR156 family. The right panel shows the gradient of expression revealed upon removal of the stele (blue box). Snapshot of the SPL6 miR156 translatome in WT and hyl1 backgrounds. Removal of the stele (blue box) reveals subtleties of miR156-mediated gene regulation in the other layers, resulting in a near-mirror image of (A).
Figure 4(A) Representative images of chromosome spreads from hSgo1 RNAi treated cells stably expressing the indicated YFP-hSgo1 variants. All the YFP-hSgo1 rescue constructs (green) localize to the centromeres (CREST, red). Scale bar, 5μm. (B) The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs from the chromosome spreads in (A) and averaged for a single cell. The data are from 3 independent experiments and the mean and SD are indicated. (C) IP of YFP-hSgo1 from cells stably expressing hSgo1 WT, 3A, and 4A followed by immunoblotting of cohesin components, Smc1 and Smc3. Representative blots are shown (top). Signals were quantification by LiCor, normalized to YFP and plotted (bottom). Error bars represent SD (n=4 for Smc1 and n=3 for Smc3). (D) Table summarizing the Log2 differences between hSgo1 WT, 3A and 4A from quantitative MS analysis. (E) Experimental protocol of the live cell imaging shown in (F). (F) hSgo1 RNAi and rescue with the indicated hSgo1 RNAi-resistant constructs was performed. Representative still images captured during the live cell imaging showing DIC and YFP-hSgo1 WT, 3A, and 4A localization during mitosis. Time (min) from nuclear envelop breakdown (NEBD) is indicated. Scale bar, 15μm. (G) The time from NEBD to anaphase was measured from 3 independent live cell imaging experiments. Each circle represents an individual cell, and the median is indicated. Note that the WAPL KD condition is incorporated from the experiment which is shown in full in Fig. EV4C for clarity.
Figure 1Figure 1. SPOP is composed of three distinct domains. (A) Cartoon schematic of the SPOP self-association incompetent mutant constructs that form complexes through only one (or neither) of the self-association domains is shown. The previously described mutation Y353E (van Geersdaele et al, 2013) was introduced into the BACK domain so that self-association could only occur through BTB-mediated interactions resulting in SPOP mutBACK; previously described mutations of the conserved BTB interface (Zhuang et al, 2009) resulted in SPOP mutBTB, which self-associates only through BACK-mediated interactions. Constructs for expression in mammalian cells encode the full length protein; those for expression in bacteria comprise residues 28-359. (B) Ribbon diagram of SPOP ΔBACK (PDB ID 3HQI (Zhuang et al, 2009)) showing residues that are mutated to abolish BTB domain self-association (L186D, L190D, L193D, I217K). The domains are colored as in Fig 1A (MATH, green; BTB, red; and BACK, blue), with one monomer shown in gray for clarity. Introduction of these mutations does not abolish the interaction between SPOP and the CRL because they are not part of the SPOP/cullin-3 interface (Zhuang et al, 2009; Errington et al, 2012) and because mutation of the BTB domain or deletion of the BACK domain does not prevent SPOP from promoting ubiquitination of substrates (Zhuang et al, 2009; Errington et al, 2012). (C) Ribbon diagram of SPOP270-374 (L273D L282D L285K) dimer crystal structure (PDB ID 4HS2 (van Geersdaele et al, 2013)) showing residue Y353, which is mutated to E to prevent BACK domain self-association. The domains are colored as in Fig 1A (MATH, green; BTB, red; and BACK, blue), with one monomer shown in gray for clarity.
Figure 7AP2-O3 protein expression from female gametocyte, female gamete, zygote, to retort stage of the ap2-o3::6HA strain. The activated gametocytes were collected at 30, 60, and 120 min and co-stained with HA and P28 antibodies. Pie charts indicate the percentage of AP2-O3+ and AP2-O3- cells. "n" is the number of cells analyzed at each time point. Western blot of AP2-O3 protein expression in parasites Diagram of CRISPR/cas9-mediated promoter swap in the ap2-o3::6HA. The promoter (467 bp) of ap2-o3 gene was replaced with the ccp2 gene promoter (1202 bp), generating the Pccp2 mutant to drive gene expression post-fertilization. Western blot of AP2-O3 protein expression in non-activated (NAG) and activated gametocytes (AG) of ap2-o3::6HA and Pccp2 strains. IFA of AP2-O3 protein expression in activated gametocytes of Pccp2 strains. Gametocyte formation in mouse. In vitro exflagellation rate of male gametocytes. Female gamete fertilization to zygote analysis by co-staining of P28 and GAP45. Mature ookinete formation in vitro. Oocyst counts in the mosquitoes at day 7 post blood-feeding. x/y on the top is the number of mosquitoes containing oocyst/the number of mosquitoes dissected; the percentage number is the mosquito infection prevalence.
Unipolar insertion of PG.Figure 5Short pulse labeling of exponential phase bacteria with the fluorescent D-amino acid HADA. Merge is showing old pole marker PdhS-mCherry (red) and PG insertion sites (white). Scale bars: 2 μm. Demographic representation of HADA labeling. Bacteria were sorted according to their cell length and oriented with the old pole at the bottom of the demograph by PdhS-mCherry fluorescence signal. NFI: normalized fluorescence intensity. n = 393 bacteria (3 biological replicates). Quantification of localization of PG insertion sites from (A). Numbers are mean percentages. n = 393 bacteria (3 biological replicates).
Figure 7(A) In full induction conditions (high galactose), bursting of the GAL genes is determined by the binding dynamics of Gal4. A longer Gal4 dwell time results in a larger burst size. The Gal4 dwell time is determined by the affinity of the UAS and is significantly reduced by fragile nucleosomes. (B) In lower galactose concentration, Gal80 represses Gal4 activity and transcription initiation during some bursts, resulting in a lower burst frequency. When Gal4 is not repressed, the burst duration is similar to full induction conditions. We propose that galactose concentration determines the availability of active Gal4. (C) In inactive conditions, Gal4 still binds DNA, but is fully repressed by Gal80.
a) Electron micrographs of fractions enriched in autophagosomes and autophagolysosomes isolated from liver of 18Q-htt and 111Q-htt mice.figf4(a) Electron micrographs of fractions enriched in autophagosomes and autophagolysosomes isolated from liver of 18Q-htt and 111Q-htt mice. Insets: higher magnification of single vesicles. Right: percentage of vesicles with electron-clear (light), vesiculated (multivesicular) or electron-dense (dark) contents. Mean + s.e.m. of three different isolations (> ∼1,000 autophagic vacuoles). (b) Bidimensional electrophoresis and Sypro Ruby staining of these fractions. Left, intact vacuoles; right, vacuole contents. Horizontal dimension, isoelectric point (pH); vertical, molecular mass (kDa). *P 0.05.
CCCP-induced Parkin translocation to mitochondria is impaired in tau expressing cells.Figure 2A Illustration of the image analysis process. The inverted "Union Jack" look-up table (LUT) was used to better visualise GFP-Parkin clusters, which were then manually selected as regions of interest (ROIs) and the corresponding mitochondrial fluorescence intensity was measured. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm. B Representative images of N2a cells expressing GFP-Parkin and hTau, hP301L or empty vector (Control) for 24 h, that were then treated with 10 μM CCCP for 4 h. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm. C Quantification of Parkin clusters/cell at 24 h post-transfection. Data were analysed with a Kruskal-Wallis test, χ2 (2) = 8.18, p = 0.0168, followed by Dunn's post-hoc analysis, n = 33-38 cells/group. D Quantification of Parkin clusters/cell at 48 h post-transfection. Data were analysed with a Kruskal-Wallis test, χ2 (2) = 19.2, p < 0.0001, followed by Dunn's post-hoc analysis, n = 37-55 cells/group cells/group. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm. E To assess colocalisation of Parkin clusters and mitochondria at 24 h post-transfection, Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment were calculated. Control r = 0.616, p = 0.0001; hTau r = 0.217, p = 0.2771; hP301L r = 0.548, p = 0.0014 (n = 27-34 cells/ group). F Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment at 48 h post-transfection. Control r = 0.434, p = 0.0167; hTau r = 0.128, p = 0.4941; hP301L r = -0.299, p = 0.2794 (n = 15-31 cells/ group). Note the different intensity scales between (E) and (F) reflect different bit depths of the images. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm.
CDC45-dependent generation of replication barriers compromise the fitness of Chk1-deficient cells.Figure 8Western Blot of CDC45 and Chk1 in U2OS cells after an extraction with CSK buffer to separate the insoluble and soluble fractions. Roscovitine (25μM) was added 2 h before harvesting. H2B was used as a loading control. IdU track lengths from U2OS cells. 200 DNA fibers obtained from 2 independent experiments were measured for each condition. IdU track lengths from U2OS cells. >170 DNA fibers obtained from 2 independent experiments were measured for each condition. For comparison, the data showing that Polη is required for roscovitine-dependent rescue of fork elongation in Chk1-depleted cells (Fig 3B) is shown on the right. The lower panel shows the corresponding Western Blot of Chk1, Polη and CDC45. Actin was used as a loading control. Percentage of origin firing (mean+SD) from DNA fibers in C (n=2). >490 fibers per condition were scored. Sensitivity of U2OS cells to Chk1 and CDC45 depletion. Cell number was determined 7 days after transfection (5 days after maximal downregulation). Data represent the mean (+SD) of 2 independent experiments. Representative image of data shown in (E). Scale bar: 100 μm. Sensitivity of U2OS cells to Chk1 inhibition and roscovitine (5 μM). Cell number was determined 4 days after a 24 h treatment (5 days in total) with Chk1i±Rosc. Data represent the mean (+SD) of 2 independent experiments. Representative image of data shown in (G). Scale bar: 100 μm.
Figure 5A Schematic view of canonical and variant H2A used for octamer assembly analysis. The H2A.B, H2A, and H2A.Z.2.2 sequences are indicated by gray, blue, and orange, respectively. The regulating-octamer-folding (ROF) sequences from different H2A histones are displayed for comparison. B Effect of the canonical and noncanonical H2A ROF on histone octamer assembly. Left: Gel filtration chromatography profiles of octamers assembled by core histones including various H2A family members or mutants. Highlighted in blue shadow are chromatography fractions subject to SDS-PAGE analysis. Right: SDS-PAGE analysis of histone octamer integrity by visualizing histone stoichiometry. The stars indicate lnkH2B-H2A containing different H2A family members or mutants present in chromatography fractions. The triangles indicate the chromatography fractions containing the majority of the lnkH2B-H2A or their mutants. C Close-up view of structure of H2A C-terminal regions shown in cartoon representation. Histones H2A, H3 and ROF region in H2A-NCP (PDB: 3LZ0) are colored in yellow, blue and red, respectively. D,E Effects of the canonical and noncanonical H2A ROF on nucleosome resistance to MNase digestion. Displayed are Native-PAGE results (D) and graph of MNase digestion efficiency calculated from quantitation of the digested DNA against total DNA (E). All experiments are repeated twice. Data are mean ± SD, n = 2.
Comparison of the simulations of the kinetic model version 1 and version 2 with experimental data of the different strains under anaerobic growth conditions.Figure 5A) Comparison of the simulations of the kinetic model version 1 and version 2 with the measured exchange fluxes. Data information: For the measurements, the means for n = 3 biologically independent samples are shown and the error bars represent ± SD. B) Comparison of the simulations of the kinetic model version 1 and version 2 with the measured metabolite concentrations. Data information: For the measurements, the means for n = 3 biologically independent samples are shown and the error bars represent ± SD. C) Metabolic map containing most relevant reactions (blue) and metabolites (black) of E. coli under anaerobic conditions. Dashed arrows indicate (lumped) reactions with several enzymes involved. Allosteric regulations of the respective reactions are marked in red (inhibition) or green (activation). The kinetic model (version 1 and 2) covers almost all of the shown reactions and regulations; a map directly related to the model is shown in Appendix Figure S1. Abbreviations of metabolites and reaction names: Glcex: external glucose (substrate); G6P: d-glucose-6-phos­phate; F6P: d-fructose-6-phosphate; FBP: fructose-1,6-bisphosphate; GAP: d-glyceraldehyde-3-phosphate; DHAP: dihydroxy­acetone phosphate; BPG: 1,3-bisphospho-d-glycerate; 3PG: 3-phosphoglycerate; PEP: phosphoenol-pyruvate; PYR: pyruvate; AcCoA: acetyl coenzyme A; CoA: coenzyme A; AKG: α-ketoglutarate; OAA: oxaloacetate; FUM: fumarate; SUC: succinate; FOR: formate; LAC: lactate; ACE: acetate; ETH: ethanol; ATP: adenosine triphosphate; ADP: adenosine diphosphate; NAD: oxidized nicotin­amide adenine dinucleotide; NADP: oxidized nicotinamide adenine dinucleotide phosphate; NADH: reduced nicotin­amide adenine dinucleotide; NADPH: reduced nicotinamide adenine dinucleotide phosphate; CO2: carbon dioxide; MQH2: mena­quinol; MQH: menaquinone. PTS: phoshotransferase system; PGI: glucose-6-phosphate isomerase; PFK: phospho­fructo­kinase; FBA: fructose-bisphosphate aldolase; TPI: triose-phosphate isomerase; GHD: glyceraldehyde-3-phosphate dehy­dro­genase; PGK: phosphoglycerate kinase; PGM: phosphoglycerate mutase; ENO: enolase; PYK: pyruvate kinase; PFL: pyruvate formate-lyase (also known as formate acteyltransferase); LDH: lactate dehydrogenase; PTA: phosphate acetyltransferase; ACK: acetate kinase; ACDH: acetaldehyde-CoA dehydrogenase; ADH: alcohol dehydrogenase; PCK: phosphoenolpyruvate carboxy­kinase; PPC: phosphoenolpyruvate carboxylase; CS: citrate synthase; ACO: aconitate hydratase A/B; ICDH: isocitrate dehydrogenase; MDH: malate dehydrogenase; FHD: fumarase; FRD: fumarate reductase; NDH: NADH dehydrogenase; ADK: adenylate kinase; NGAM: ATP consumption for non-growth associated maintenance; ATPase: ATP hydrolysis by F1-ATPase in the ATPase strains.
Figure 2(A) Immunoblot analysis of AGIA-PLZF protein levels in AGIA-PLZF and FLAG-CRBN expressing HEK293T cells treated with DMSO or lenalidomide (Len) in the presence of DMSO, MG132 or MLN4924 for 9 h. (B) Immunoblot analysis of endogenous PLZF protein levels in HEK293T cells or CRBN-/- HEK293T cells treated with DMSO or lenalidomide (Len) for 24 h. (C) Immunoprecipitation of FLAG-CRBN in FLAG-CRBN and AGIA-PLZF expressing HEK293T cells treated with DMSO or lenalidomide (Len) in the presence of MG132 for 8 h. Components of CRL4FLAG-CRBN and AGIA-PLZF were detected using each specific antibody, as indicated. (D) Ubiquitination of AGIA-PLZF in AGIA-PLZF and FLAG-CRBN expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) in the presence of MG132 for 10 h. AGIA-PLZF was immunoprecipitated using anti-AGIA antibody and the polyubiquitin chain on AGIA-PLZF was analysed by immunoblot. (E) In vitro binding and ubiquitination assay of AGIA-PLZF. Empty vector, AGIA-PLZF or FLAG-CRBN expressing HEK293T cells were lysed and the lysates were mixed. The first immunoprecipitation with anti-AGIA or anti-FLAG antibodies was performed in the presence of DMSO or 200 µM lenalidomide. The purified AGIA-PLZF or CRL4FLAG-CRBN complex, including AGIA-PLZF and FLAG-CRBN, was incubated with recombinant E1, E2 and HA-ubiquitin in the presence of DMSO or 200 µM lenalidomide. The second immunoprecipitation was performed using anti-AGIA antibody. Ubiquitination of PLZF was analysed by immunoblot.
a) Representative merged images using GFP–LC3 MCF-7 cells expressing Flag–TSC1, Flag–TSC2 or both Flag–TSC1 and Flag–TSC2 showing GFP–LC3 (green) puncta.figf4(a) Representative merged images using GFP-LC3 MCF-7 cells expressing Flag-TSC1, Flag-TSC2 or both Flag-TSC1 and Flag-TSC2 showing GFP-LC3 (green) puncta. Scale bars, 10 μm. (b) Quantification of GFP-LC3 puncta was performed and the results are represented as the average puncta fluorescence per cell (±s.e.m., n = 3 independent experiments) from 100 cells per experiment as shown in a. ***P0.001; NS, not significant. (c) Representative immunocytochemistry images using MCF-7 cells transfected with an mRFP-GFP-LC3 construct. Cells treated with 0.4 mM H2O2 were analysed at 0 h (Control), 1 h, 3 h and 6 h. Scale bars, 10 μm. (d) Quantification of autophagosomes (AP, GFP-LC3) and autolysosomes (AL, RFP-LC3) per cell in different conditions as shown in Fig. 4c. The results are represented as the average puncta fluorescence per cell (±s.e.m., n = 3 independent experiments) from 100 cells per experiment. **P0.01;***P0.001. (e) Western analysis of FAO cells treated with 50 μM Wy-14643 (WY) or vehicle (DMSO) for PPAR-alpha-inducible proteins (EHHADH and ACAA1), mTORC1 signalling proteins ((pS6 (S235/236), S6, pS6K (T389) and S6K)) and autophagy markers (LC3 and p62). (f) Representative images of FAO cells treated with vehicle (DMSO) or 50 μM Wy-14643 (WY) for 1 h, with superoxide production detected using dihydroethidium (DHE). Scale bars, 30 μm. (g) Representative images of LC3 puncta (green) in FAO cells following treatment with 50 μM Wy-14643 (WY) or vehicle (DMSO) for the indicated time periods. Scale bars, 10 μm. (h) Quantification of LC3 puncta per cell in response to Wy-14643 (WY) or vehicle (DMSO). The results are represented as the average LC3 puncta fluorescence per cell (±s.e.m., n = 3 independent experiments) from 100 cells per experiment as shown in Fig. 4a. *P0.05; **P0.01. (i) Transmission electron microscopy of FAO cells treated with vehicle (DMSO) or 50 μM Wy-14643 (WY) for 24, 48 and 72 h. Peroxisomes and autophagosomes are indicated with red and yellow arrows, respectively. Scale bars, 500 nm. Uncropped images of western blots are shown in Supplementary Fig. S6. Source data for the statistical analysis are shown in Supplementary Table S1.
Figure 4Gene ontology (GO) analysis of biological processes for transcriptionally upregulated genes (≥1.5-fold; adjusted p-value<0.05) after combined VPS4A+B silencing using the enrichGO function from clusterProfiler. Heatmap visualizing expression of genes related to inflammatory response (left panel) and positive regulation of cell death (right panel) generated from the GO analysis of biological processes across different transfection conditions with at least 3 biological replicates. Selected pathways related to inflammatory response and programmed cell death among transcriptionally upregulated genes after combined VPS4A+B silencing were identified using the enrichPathway function from ReactomePA.
a) HEK293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated in the presence of amino acids.figf5(a) HEK293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated in the presence of amino acids. Lysates were immunoblotted with the indicated antibodies. A representative experiment of three repeats is shown. (b) UVRAG bridges the interaction between Beclin-1 and ULK1. HEK293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated. Lysates were immunoprecipitated with anti-HA(Beclin-1) antibody and blotted with the indicated antibodies. A representative experiment of three repeats is shown. Uncropped images of blots are shown in Supplementary Fig. S4.
The schematic of teicoplanin blocking the entry of 2019-nCoV.Figure 4. The schematic of teicoplanin blocking the entry of 2019-nCoV.Schema graph of the 2019-nCoV for entry into target cells. After binding of virus to the cellular receptor ACE2, the proteolytic process was initialed by TMPRSS2 on the cellular membrane. The virions will be up-taken into endosomes, where the S protein is further activated by cleavage with cysteine protease cathepsin L. The cleavage of S protein by cathepsin L can be significantly blocked by teicoplanin.