EMBO/sd-character-level-ner huggingface dataset:
CCB02 enhances microtubule nucleation activities of centrosomes (A) Experimental scheme of microtubule (MT) regrowth assay.Figure 6(A) Experimental scheme of microtubule (MT) regrowth assay (B-D) MT-regrowth assays at 0, 1.5 and 3.0 min using MCF10A (-Dox, two centrosomes), MCF10A (+Dox, extra centrosomes) and MDA-MB-231 cells. MT nucleation panel is shown in grey scale (inset images are inverted) and γ-tubulin is shown in red. Note that in contrast to vehicle treatment, CCB02 treatment caused centrosomes to nucleate an enhanced level of microtubules already at 1.5 min after induction of regrowth with simultaneous increase in γ-tubulin recruitment. All these cells were stained with γ-tubulin (red), microtubules (α-tubulin, grey) and DNA (DAPI blue). Scale bar, 2µm and insets, 0.5 µm. Bar graphs show MT and γ-tubulin intensities at 3 min after induction of MT regrowth. (N)=3. At least 80 centrosomes were considered to calculate intensities from each cell line. Error bars, mean ± SEM. Unpaired t-test **P < 0.001, ***P < 0.0001.
Cryo-EM results of SARS-CoV-2.Figure 3.(A) and (B). Cryo-EM images of SARS-CoV-2. (C). Zoom-in view of the virion showed in (A). Envelope and nucleocapsid are indicated by green and blue respectively, remarkable spikes are indicated by red triangles. (D). Zoom-in views of the two virions showed in (B), remarkable spikes are indicated by red triangles. (E). Zoom-in view of the spike indicated by yellow triangle in (C). The shape is depicted by yellow dot lines. (F). Zoom-in view of the spike indicated by yellow triangle in (D). The shape is depicted by yellow dot lines.
Figure 4A. Gcn5 binding to chromosomes assessed by chromosome spreading. Fission yeast nda3-KM311 cells were shifted at 19°C for 4 hours to enrich the population for the early stages of mitosis, and processed for chromosome spreading and immunofluorescence microscopy against Gcn5-myc and Cut14-HA. The Cut14/DAPI ratio served as marker for mitotic progression, with interphase (i): ratio<0.5; prophase (p): 0.51.5. For each strain, four representative nuclei extracted from a same image acquired with same settings are shown. Bar: 5 µm. Boxes indicate the 25th, median and 75th percentile and whiskers the min and max values (n>150 nuclei per strain). B. ChIP against Gcn5-myc on cycling cells or cells arrested in pro/metaphase. Mitotic indexes in parentheses were determined by scoring binucleated cells and hypercondensed nuclei. % IP are averages and s.d. calculated from 6 ChIPs on 3 biological replicates. C. Total H3 and acetylated isoforms in cycling or mitotically arrested cells. Whole cell extracts (WCE) prepared from cycling cells or prometaphase cells (mitotic index 89%) were loaded on a same gel and simultaneously revealed. D-E. Chromosomal occupancy of acetylated H3 during mitosis. Cells expressing Cnd2-GFP were blocked in early mitosis, mitotic indexes measured (D), and processed for ChIP against total H3, H3K9ac or H3K18ac. Heterochromatin (dh1) served as negative control. H3ac/H3 average ratios and s.d. calculated from 3 ChIPs on 3 biological replicates are shown. *** P<0.001, * P<0.05, °P>0.05. High-occup. and Low-occup. refer to condensin high-occupancy and low-occupancy binding sites, respectively.
Fig.Figure 2A, Scheme of HoxA and HoxB. The open reading frame of hoxA is interrupted by one 67 bp long intron (RNAseq data) and encodes a protein of 380 amino acids with a predicted mass of 42.5 kDa. The protein contains a heme binding pocket, a HemeO-like superfamily domain and a C-terminal putative membrane anchor (CTA). In comparison, hoxB is interrupted by a 50 bp intron. The protein (509 amino acids, 55.9 kDa) contains only the HemeO-like superfamily domain. B, Effect of the deletion of hoxA or hoxB on red-light induction of the ccgA gene. A. alternata strains were grown for 36 h at 28°C. Samples were illuminated for 1 h in red light while controls were kept in the dark. The amount of ccgA transcript was quantified by qRT-PCR analyses with the histone 2B gene as housekeeping gene. Error bars represent the standard deviation (n = 3). C, Comparison of wild-type colonies with phytochrome and hox-deletion strains in the presence of 0.12 mM menadion (MD), 7 mM H2O2 or 2 mM tert-butyl hydroperoxide (tBooH). The strains were incubated for 5 days at 28°C in the dark.
Aster-C deficiency leads to hyper-activation of mTORC1.Figure 1A. Western blot analysis of mTORC1 activity in C2C12-Vevtor (VC) and Aster-C KO (KO) cells in complete medium (CM) and in response to nutrient starvation or stimulation with amino acids for 30 min. B and C. Statistical analysis of phosphorylated level of S6K (B) and 4E-BP1 (C) in C2C12-Vector and Aster-C KO cells in CM, under nutrient starvation (-AA) and in response to stimulation with amino acids (+AA) using ImageJ software. Data are represented as mean ± SD (n=3 biological replicates). *p < 0.05, **p<0.01 by Student's t test. D. Immunostaining of endogenous mTOR and LAMP1 in C2C12-Vector and Aster-C KO cells under nutrient starvation (-AA) and in response to AA stimulation (+AA). LAMP1 was immunostained as a lysosome marker. Arrows highlight the co-localization of mTOR with LAMP1. Scale bar, 40 μm (Row 1) and 10 μm (Rows 2, 3, 4). E. Statistical analysis of the Pearson's correlation coefficient of mTOR and LAMP1 in Figure 1F (n=12-15 cells per group). Data are represented as mean ± SD. ***p<0.001 by one way ANOVA. F. Western blot analysis of mTORC1 activity in Aster-C KO cells stably re-expressing exogenous GFP-Aster-C under nutrient starvation and AA stimulation. Aster-C KO cells were transfected with GFP-Aster-C and selected with G418 for 2 weeks. G. Immunostaining of endogenous mTOR and LAMP1 in C2C12-Vector and Aster-C KO cells under cholesterol depletion by treatment with 0.5% MCD for 2 hrs (-CHOL) or cholesterol (50 µM) re-stimulation for 1 hr (+CHOL). Arrows highlight the co-localization of mTOR with LAMP1. Scale bar, 40 μm (Row 1) and 10 μm (Rows 2, 3, 4). H. Western blot analysis of mTORC1 activity in C2C12-Vector and Aster-C KO cells in CM, under starvation, cholesterol depletion by treatment with 0.5% MCD for 2 hrs or cholesterol (50 µM) re-stimulation for the indicated time.
Fig.Figure 4A. Scheme of the different steps of RNAPII regulation for which the corresponding rates were modelled B, C. Boxplots reporting LPS-induced changes (Log2FC) for each of the four RNAPII kinetic rates in c-mycwt/wt (wt) and c-myc∆/∆ cells for Myc-dependent (B) and Myc-independent (C) induced and repressed genes. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported. The RNAPII ChIP-seq time-course was performed on two biological replicates (each on pools of cells from > 10 mice), and reads were merged after sequencing. D, E. Heatmaps showing the variations in RNAPII kinetic rates (Log2FC) on Myc-dependent (D) and Myc-independent (E) genes during the LPS time-course. The grey scale represents the starting level for each parameter in unstimulated cells. The first column on the left reports the changes in Myc share for the same genes. Genes were grouped using hierarchical clustering. F. Boxplots reporting LPS-induced changes (Log2FC) for each of the four RNAPII kinetic rates in c-mycwt/wt (wt) and c-myc∆/∆ cells for genes of clusters 1 and 9. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported. G. Changes in Myc share in LPS treated versus untreated cells for the different clusters of panel d. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported.
Mutations in the LBD of DHR3.Figure 5(A, B) The ap-Gal4 driver induces dS6K in combination with either (A) the DHR3-EP transgene or (B) the EP with the EMS DHR3 mutation (C) Structure of the full-length DHR3-PA protein showing previously described DBD mutants (G60S, R107G) and the EMS mutants that lack the LBD (K243X, W284X); the light and dark grey boxes represent the DBD and the LBD respectively. (D) dS6K activity in larval protein extracts measured by H2B target phosphorylation and (E) corresponding western blot of dS6K in the trans-heterozygote LBD-mutant combination (LBD), as compared to heterozygous larvae (Co). (F, G) The eyeless promoter directs the flipase recombinase during eye development. Flipase allows recombination on the right arm of the second chromosome and eventually leads to an adult eye (F) that is homozygous for the wild-type allele of DHR3. (G) Homozygosity for a DHR3K243X mutation results in a decrease in the size of the adult eye. A dotted yellow line surrounding the control eye in (F) has been copied and pasted on the mutant eyes (G); insets are higher magnification images of ommatidia showing misorientation of bristles. (H) At the scutellum, a y-marked homozygous DHR3K243X mutant bristle (arrow), recognizable by its light color, is smaller than the symmetrical neighboring bristles.
Summary Workflow.Figure 4 We began by identifying publicly available datasets from transcriptome profiling experiments that analyzed blood leukocyte samples from adult and child sepsis patients. After data processing, we used Pathprint to translate these gene expression patterns to the pathway activity level. By comparing samples at the pathway level the Pathprint method is robust to batch effect and allows for comparison across multiple array platforms. After identifying age-associated differences in pathway activity, we used them to facilitate drug discovery by constructing targeted Pathway Drug Networks (PDNs). This novel method works by incorporating our target pathways into a base network built upon the correlation in the expression of >16,000 disease, pathway, and drug gene signatures across >50,000 individual microarrays. The resultant network neighborhood was used to identify drugs with positive or negative association with high-survival (child) or high-mortality (adult) pathways, respectively. We validated top drug leads by curating and analyzing prior data collected in pre-clinical models of sepsis and also by directly testing their ability to improve survival in a mouse model of fatal endotoxemia
Figure 1A, A targeted MAPPIT screen identifies several DUBs as putative RAS interactors. A MAPPIT array containing DUB prey library was screened with HRAS G12V as bait. pSEL(+2L)-HRAS G12V was expressed in HEK293T cells together with the indicated prey. BRAF served as a positive control. Each measurement was done in triplicate. The results are expressed as a mean of normalized luciferase activity (leptin-treated cells vs leptin-untreated cells). The overall mean value + 2 s.d. served as a threshold. B, MAPPIT assay confirms the interaction between OTUB1 and RAS proteins. pSEL(+2L) vectors coding RAS proteins were expressed in HEK293T cells together with the indicated prey. Empty vector and two random baits, MAL and eDHFR, were used as negative controls. REM2 and EFHA1 preys that bind to the bait receptor itself was used to evaluate expression of the RAS baits. The results are expressed as a mean of normalized luciferase activity ± s.e.m (leptin-treated cells vs leptin-untreated cells), n=3. C, NRAS Q61K mutant co-immunoprecipitates with OTUB1. At 48 hours post-transfection with Flag-tagged NRAS Q61K and HA-tagged OTUB1 expression constructs, HA-tagged OTUB1 was immunoprecipitated with anti-HA-agarose followed by immunoblotting using anti-Flag or anti-HA antibodies. D, OTUB1 interacts with wt NRAS and active NRAS mutant. Flag-tagged NRAS proteins were immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing HA-tagged OTUB1 or empty vector (V). E, GTP binding does not affect the complex formation between NRAS and OTUB1. Recombinant Flag-tagged NRAS was incubated with lysates derived from HEK293T cells expressing HA-tagged OTUB1 in the excess of GTP-γ-S or GDP, followed by immunoblotting with the indicated antibodies. F, OTUB1 interacts with wt KRAS. Flag-tagged KRAS was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing HA-tagged OTUB1 or empty vector (V). C, D, E, F, Immunoprecipitates, IP. Whole cell lysate, WCL. G, OTUB1 co-localizes with KRAS at the plasmamembrane. At 24 hours after co-transfection with GFP-tagged KRAS and HA-tagged OTUB1, HeLa cells were immunostained with anti-HA antibody. The outlined areas are shown at higher magnification at the top of each image. Scale bar, 10 micron.
Figure 6(A-D) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of Smc5/6 complex (35oC) in smc6-9 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of Smc5/6 complex (mean ± SD from two independent experiments). (E) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of Smc5/6 complex.
Figure 2A Mitochondrial translation products were labeled in WT HeLa cells for indicated time lengths (heat map) and cells immunostained for TOM20 (gray). Representative confocal images are shown for each time point. The same 5-minute image was used in both rows with different brightness as a reference for intensity comparison of the other time points in the row. Scale bars, 10 µm. Mitochondrial protein synthesis was quantified over time. 30 cells from N = 3 independent experiments were analyzed for each time point (Average of the HPG intensity in the mitochondrial area was calculated and the baseline was set to zero by subtracting the value of time point 0 from all the values. Error bars, +/- SEM). A hyperbolic graph was fitted to the data. R-squared, 0.97. B As in A, Mitochondrial translation products were labeled for increasing time (10 and 20 minutes) in HeLa cells. The number of analyzed cells was increased in the plots from 25 to 300 per time point. 150 or 300 random cells from N = 3 independent experiments were analyzed. Kruskal-Wallis followed by Fisher's test. ****p < 0.0001. Error bars, +/- SEM. C Mitochondrial translation products were labeled for 30 minutes and clicked with AF647 (magenta) and cells immunostained for TOM20 (green) in fibroblasts, iPSCs-derived cardiomyocytes, and hippocampal neurons. Representative epifluorescent microscopy images are shown. The correlation of HPG and TOM20 channels is represented in fire lookup table, after calculating the Pearson Correlation values over each pixel of the images. Scale bars, 25 µm.
Figure 6A-B. Co-immunoprecipitation assay. The solubilized lysates from 2×1010 EAEC wild-type or ∆mltE cells producing FLAG-tagged TssM (FLTssM) and/or HA-tagged TssJ (TssJHA, panel A) or TssL (TssLHA, panel B) (Total, T) were subjected to immune precipitation on anti-FLAG-coupled agarose beads. The immunoprecipitated material (IP) was subjected to 12.5%-acrylamide SDS-PAGE and immunodetected with anti-FLAG (upper panel, TssM) and anti-HA (lower panel, TssJ or TssL) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiments were performed in triplicate and a representative result is shown. C. BS3 cross-linking assay. 2×109 cells of the indicated strain producing FLAG-tagged TssM (with the exception of ∆tssM cells) were treated (+) or not (-) with the BS3 cross-linker agent. After the cross-linking reaction, cells were boiled in Laemmli buffer and total proteins were subjected to 7%-acrylamide SDS-PAGE and immunodetected with anti-FLAG antibodies. The TssM protein (FLTssM) and its complexes (*, TssM-TssJ; **, TssM-TssL) are indicated on the right as well as the TssM dimer (arrow). Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown. D. Fluorescence microscopy. Recordings showing TssM localization using the chromosomally-encoded sfGFP-tssM fusion in wild-type (WT) or ΔmltE cells or ΔmltE cells producing the wild-type (mltE+) or catalytic variant (mltEE64Q) MltE protein. Scale bars are 1 μm. The experiment was performed in triplicate and a representative result is shown.
GOT2 acetylation promotes tumor growth, and GOT2 K159 acetylation is increased in pancreatic cancer A–C GOT2 3K acetylation promotes xenograft tumor growth.Figure 5A-C GOT2 3K acetylation promotes xenograft tumorgrowth. Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins (2 × 106cells) were injected subcutaneously into the flanks of nude mice. The tumor volume was carefully monitored over the indicated time period (A). At 8 weeks after injection, tumors from six mice were extracted, photographed, and weighted (B, C). D GOT2 3K acetylation promotes tumor cellproliferation. Tumor sections from xenografts were prepared for IHC staining, and the PCNA‐stained tumor sections were analyzed by quantifying the PCNA‐positive area. E Clinical cases with increased GOT2 K159 acetylation in humanpancreatictumor tissues. 10 pairs of humanpancreatictumor tissue (shown as T) and adjacent normal tissue (shown as N) were lysed. The acetylation level of GOT2 at K159 was compared against GOT2protein by Western blot.F Clinical cases with decreased SIRT3protein expression in humanpancreatictumor tissues. 10 pairs of humanpancreatictumor tissue (shown as T) and adjacent normal tissue (shown as N) were lysed. The SIRT3protein levels were compared against tubulin by Western blot.G, H Quantification of GOT2 K159 acetylation and SIRT3protein expression in clinical cases as described in (E) and (F).
Figure 5Thermal shift assays were used to determine the melting curves of wild-type CggR and mutants. Error bars correspond to the standard deviation of at least five replicates. CggR-FBP affinity constants (KD's) of the wildtype and mutant variants, determined by fitting a simple cooperative binding model to the melting curves data. Error bars indicate the 95% confidence intervals. Quantification of CggR binding to DNA. The CggR bound to DNA fraction was calculated by dividing the intensity of the protein-DNA complex band by the total DNA. The background-subtracted total intensities of the CggR-DNA complex and the free-DNA bands were assessed with ImageJ. Error bars correspond to the standard deviation of three replicate experiments for the mutants and six for the wildtype. Summary of the biochemical characterization of the wildtype and mutant CggR variants. KD ratio change indicates the ratio between the KD of the CggR mutant variant and the one of the wildtype. A plus sign indicates desired characteristics achieved in the mutants; a minus sign indicates undesired effects of the mutations. The mutant highlighted with grey background is the one we selected for further analyses, as this mutant had the desired characteristics with regards to all three criteria. Ray-traced picture of the wildtype CggR and R250A mutant structure. Carbon atoms of the FBP ligand are in turquoise while the part of the side chain that is eliminated by the R250A mutation is in green. Hydrogen bonds are indicated with dashed lines.
Fig.Figure 3(A) Quantification of mtDNA levels after Southern blot analysis. Bars present mean levels ± SD (n=4). (B-C) Northern blot analysis and corresponding quantification of mitochondrial (B) rRNAs and mRNAs or (C) tRNAs. 18S rRNA is used as loading control. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (12S n=5 and the rest of samples n=4). (D) Western blot analysis of LRPPRC levels. VDAC was used as a loading control. (E) In organello transcription analysis upon 60 minutes labelling with -32P-UTP.
Genetic analysis of fifteen CUP metastases.Figure 3Somatic mutations distribution: SNVs and InDels displayed by each metastasis (listed in the Y axis) were detected by Strelka2 tool (Kim et al., 2018). Brown traits represent single mutations. The number of fully shared (red), partially shared (orange) or private (yellow) mutations is indicated in the horizontal bar below. Five cancer-associated genes (TP53, ARID2, NTRK1, SMARCA4 and ZFHX3) mutated in all metastases are highlighted. Genetic similarity among metastases: the heatmap has been drawn according to the Jaccard index. The similarity ranges from 58% to 82% among all pairs with the exception of L_08 due to the scarce number of mutations. Clonal composition analysis: representative nested view of metastasis L_01. The five sub-clones (represented in different colours) inferred by clustering similarities of the variant allele frequencies (VAFs) follow a linear pattern. Variant allele frequency of fully shared mutations present in copy neutral regions. The box plot represents the fluctuation of the VAF (in the Y axis) of single mutations (grey dots) in each metastasis; each box represents the upper and lower quartiles, while the central short black line within each box represents the median; whiskers indicate variability outside the upper and lower quartiles. Reconstruction of the phylogenetic tree linking the fifteen CUP metastases: the model was reconstructed by taking into account the maximum likelihood molecular evolutionary tree according to Phylip (Felsenstein, 2005) and the incremental number of mutations. 'Brooks' lengths are proportional to the amount of 'private' mutations.
Figure 6 Spot assay of designed TAD constructs . The spot assay was performed at 30ºC (left) and during heat shock at 37ºC (right panel)
dependent cellular auxin efflux.Figure 1A Normalized firefly luciferase luminescence (compared to renilla luciferase) as a function of external IAA concentration. Arabidopsis protoplast cells were transiently transformed with either AtPIN1 or GFP (in the case of the control) both under the control of a constitutive CaMV 35S promoter. Where indicated, 10 µM NPA was added. B Normalized firefly luciferase luminescence (compared to renilla luciferase) as a function of external NPA concentration in the presence of 100nM IAA in control GFP- (blue or red) or PIN1- (green or purple) transformed protoplasts.
Figure 3Three pairs of TGF-β-treated isogenic NSCLC cell lines (H1975, HCC4006, and HCC366) and two mesenchymal NSCLC cell lines (Calu6 and H1792) were incubated with 50nM volasertib for 24 hours, lysed, and subjected to reverse phase protein array analysis. Experiments were done in triplicate. Thirty-three proteins were differentially regulated between epithelial and mesenchymal NSCLC cell lines after treatment with volasertib, including those involved in the cMet/FAK/Src signaling axis (blue text) and those involved in the PI3K/Akt signaling axis (orange text). The same cell lines treated identically were subjected to immunoblotting for the indicated proteins (upper) with densitometric quantification normalized with β-actin (lower). Epithelial (red text) and mesenchymal (blue text) NSCLC cell lines transfected with 10nM siRNA as indicated for 48 hours. NT, non-targeting control and subjected to Western blotting with the indicated antibodies. Data are means ± standard error of the mean from three independent experiments. Significant differences using two-sided Student t test are indicated by *p < 0.05. H1975 and Calu6 cells were treated with indicated inhibitors for 4 hours or 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.
Figure 7 T-bet deficient lungs infected with sub-lethal dose of influenza virus upregulate Th2 and Th17 responses, leading to increased pulmonary infiltration of neutrophils and eosinophils. This altered inflammatory response did not compromise efficiency in viral clearance or eventual tissue homeostasis, and in fact reduced tissue leakiness following infection. Furthermore, the immune environment resulting from influenza infected T-bet deficient lungs conferred immunity and survival advantage to pneumococcal superinfected hosts. Correspondingly, neutralization of IL-17 or neutrophil depletion by antibody treatment in T-bet-/- hosts reduced pulmonary neutrophil infiltration or antimicrobial peptide expression, reversing immunity against secondary bacterial infection