EMBO/sd-character-level-ner huggingface dataset:
Figure 7A. Correlation of magnitude and direction of change of genes dysregulated (relative to wild type) in timeless vs. ddx11 DT40 cells. rs (Spearman rho) is shown for each correlation. B. Genes dysregulated in timeless, ddx11 and fancj mutants have a higher density of G4s around their TSS. Cumulative fraction of the genes dysregulated in timeless (red), ddx11 (blue) and fancj (green) containing n (x-axis) G4 motifs within 1.5kb of the TSS compared with all genes (black). C. Metagene analysis showing G4 frequency around the TSS of genes dysregulated (up or down) in timeless (left panel), ddx11 (centre panel) and fancj (right panel) compared with all genes (black line). G4 frequency is calculated separately for coding (above the x-axis) and template strands (below the x-axis). D. A model for recognition and processing of replisome associated G4s by Timeless / DDX11. Current evidence suggests that Timeless is a constitutive component of the replication fork. the C terminus of Timeless may help detect G4s in the vicinity of the replisome by a combination of direct recognition through the DNA binding domain and indirectly through the PARP-binding domain. It is not currently possible to distinguish whether this mechanism would operate ahead or behind the fork itself although recent structural evidence placing Tof1, the yeast homologue of Timeless, ahead of the fork would appear to make the first possibility more probable. However, for failure to resolve G4s ahead of the fork to result in uncoupling would require the CMG helicase to traverse the structure, as has been suggested for interstrand crosslinks
vBcl-2 Interaction with Beclin1.Figure 2(A) Co-immunoprecipitation (Co-IP) of Beclin1 with WT or mutant vBcl-2. 293T cells were transiently transfected with the indicated constructs, followed by immunoprecipitation of HA-tagged vBcl-2 and immunoblotting of V5-tagged Beclin1. (B) Co-IP of WT or mutant vBcl-2 with endogenous Beclin1. 293T cells were transfected with the indicated vBcl-2 constructs, followed by immunoprecipitation of HA-tagged vBcl-2 and immunoblotting of endogenous Beclin1. 1% whole-cell lysates (WCLs) was used as the input. (C) vBcl-2 interaction with Bak protein. 293T cells were transfected with WT and mutant forms of vBcl-2 as indicated. At 48 h posttransfection, WCLs were mixed either with GST-BakΔTM fusion protein (left panel) or with GST alone (right panel) for an in vitro GST pull-down (GST PD) assays. GST fusion proteins used for the pulldown assay are indicated (bottom panel). 1% WCL was used as the input. Data are representative of at least three experiments yielding similar results.
Figure 2A Isothermal calorimetry (ITC) assays using ADP-ribose ligand and purified macrodomains of human macroH2A1.1 and macroH2A2. B In vitro PARP1 activity assay in the presence of increasing concentrations (10, 25 and 50 μM) of purified macrodomains of human macroH2A1.1, macroH2A1.1 G224E mutant and macroH2A2. PARP1 activity is assessed by its auto-PARylation detected by an anti-PAR immunoblot. The naphtol blue staining shows the increasing amounts of purified macrodomain added to each reaction. A representative blot of one of three independent experiments is shown.
Cacna1atg-la and Cacna2d2du-2J mice have autophagy defects.Fig 4A. Calbindin and hematoxylin staining of mice cerebella indicates that Cacna1atg-la mice have progressive PC and granule cell loss upon aging. Scale bar, 200 μm. . B, D. Calbindin (green) and 4',6-diamidino-2-phenylindole (DAPI)(blue) staining of micecerebellum indicates that there are swollen PC axons (arrows) in the granular layers of the Cacna1atg-lamicecerebellum at day 25 (B) and the Cacna2d2du-2Jmice at day 50 (D). Scale bar, 20 μm C, E. The TEM of the axons at the granular layers of the cerebellum showing that many PC axons in Cacna1atg-la and Cacna2d2du-2J mice are swollen and accumulated with numerous mitochondria, autophagosome/MVBs-like structures (red arrows), and various cytoplasmic vesicles or aberrant membrane structures, including stacks of cisternal membranes (blue arrows), and folded ER (yellow arrows). Scale bars for right two panels are 2 μm. Other scale bars are 500 nm F, G. LC3II and p62 are slightly increased in 15-days-old Cacna1atg-la mice and 50-days-old Cacna2d2du-2J mice. b, d are quantification of a and c. Data are presented as means ± SEM. (*: p < 0.05).
Figure 3(A and B) qRT-PCR and western blot analyses of TGFβR1 mRNA and protein levels in QKI-5-overexpressing A549 and H1299 cells. (C and D) Expression of TGFβR1 mRNA and protein in QKI-5-silenced A549 and H1299 cells. (E and F) The mRNA stability of TGFβR1 was examined in QKI-5-overexpressing/-silenced and control A549 and H1299 cells. Cells were treated with actinomycin D (Act-D, 10 μg/ml) for 0, 2, 4 or 6 h, and mRNA levels of TGFβR1 were determined by qRT-PCR. The quantity value of qRT-PCR at 0 h was set 1. (G and H) After being serum starved for 24 h, A549 and H1299 cells stably overexpressing QKI-5 were treated with or without TGF-β1 (5 or 10 ng/ml) for 24 h and subjected to western blot assays to determine the protein levels of QKI-5, TGFβR1, p-SMAD3 and SMAD3. β-actin was used as an internal control. (I and J) QKI-5-silenced A549 and H1299 cells were treated as above. Then, western blot analysis was conducted to examine the protein levels of QKI-5, TGFβR1, p-SMAD3 and SMAD3. (K) QKI-5-overexpressing A549 and H1299 cells were treated and subjected to qRT-PCR assays to detect the mRNA expression of downstream genes of the TGF-β/SMAD signaling, including PAI-1, Slug, Snail, E-cadherin, and/or N-cadherin.
Bioinformatic analysis of MitoT cells identifies changes in mRNA expression associated with T cell activation and differentiation pathways.Figure 3A Differentially expressed (DE) genes from whole transcriptome RNA sequencing analysis in FACS-sorted CD3+MitoTneg and CD3+MitoTpos cells from healthy human peripheral blood samples (n=4 biological replicates, with 4 different donors of PBMCs). B Volcano plot analysis derived from FPKM values showing DE genes (p<0.05, Student's t test) in sorted MitoTneg and MitoTpos CD3+ cells (from 4 different donors of PBMCs). C Heatmap depiction of RNA-seq analysis of significantly (p<0.01, Student's t test) DE coding genes from sorted MitoTpos and MitoTneg CD3+ cells (from 4 different donors of PBMCs). D Significantly enriched pathways related to T cell regulation and activation for DE genes (p<0.05, Student's t test) between MitoTpos and MitoTneg CD3+ cells, from the GO Biological Process 2015 database (from 4 different donors of PBMCs). E Differentially expressed genes from the significantly enriched pathways listed in figure 2D. Highlighted in red are key markers of Treg phenotype. F qRT-PCR analysis of genes related to T cell activation after mitoception with UC-MSC MT (n=4 biological replicates, with 4 different donors of PBMCs). The graph depicts fold expression in CD3+MitoTpos cells relative to CD3+MitoTneg cells, which are set at 1. Graph shows mean ± SEM and statistical analysis by Student's t-test (*p<0.05). G Correlation analysis of 14 sampled genes between qRT-PCR and RNA-seq, from MitoTpos and MitoTneg CD3+ cells (from 4 different donors of PBMCs). Graph depicts best-fit line and 95% confidence intervals by Pearson correlation analysis.
Antagonist can activate AR and provoke biological response through binding to antagonist‐liganded ARBEHeat map showing the signal intensity of AR binding in LNCaP cells treated with DHT, bicalutamide, and enzalutamide, respectively.Figure 6A Heat map showing the signal intensity of AR binding in LNCaP cells treated with DHT, bicalutamide, and enzalutamide, respectively. Numbers indicate the antagonist‐responsive AR locations in each category. B Clustering of the antagonist‐responsive AR locations based on dynamic signal shifting of H3K4me2 affected by DHT treatment. C The aggregated tag density is shown on the forward (blue) and reverse (red) strands, separately. The bottom panel represents the bound motif sequences found in the antagonist‐responsive AR locations. Antagonist‐liganded ARBE is also shown. D EMSA was performed to validate specific AR binding to antagonist‐liganded ARBEs. Biotin‐labeled wild‐type (WT) or mutated (Mut) probes were incubated with same amounts of nuclear extract from LNCaP cells treated with bicalutamide or enzalutamide. Arrow indicates the position of the shifted specific probe. Four probes (M1-M4) were randomly selected. Quantification of EMSA gel bands shows that antagonists increase the in vitro binding to 1.7-3.8‐fold compared to the vehicle. E Top panel represents overlap between genes within 20 kb of the bicalutamide‐responsive AR locations and enzalutamide‐responsive AR locations. Bottom panel indicates microarray analysis of genes around enzalutamide‐responsive AR locations following treatment with DHT or enzalutamide in LNCaP cells. F UCSC genome browser views of sequencing data at CPEB4 locus under different conditions. The blue region represents antagonist‐responsive AR location, while the two pink regions indicate agonist‐responsive AR locations. G ARChIP‐exo library validation on antagonist‐responsive and agonist‐responsive AR locations referred to (F). The data are the mean of triplicates ± SD. H mRNA levels of CPEB4 were examined in cells treated with or without DHT and enzalutamide. The data are the mean of triplicates ± SD. I Silencing of CPEB4 enhanced the inhibitory effects of enzalutamide in LNCaP cells. LNCaP cells were transfected with a control siRNA or siCPEB4. Cells were then treated with vehicle or 1 μM enzalutamide, and cell numbers were determined on day 3 with a direct viable cell count assay. The data are the mean of triplicates ± SD.
Figure 11.SARS-CoV-2 activates delayed innate immune responses in lung epithelial cells Measurements of replication and innate immune induction in Calu-3 lung epithelial cells infected with SARS-CoV-2 at MOIs 0.08, 0.4 and 2 TCID50VERO/cell. (A) Replication of SARS-CoV-2 genomic and subgenomic E RNAs (qRT-PCR). (B) Quantification of N staining from cells in (A) by flow cytometry. Mean percentage of N-positive of all live-gated cells is shown +/- SEM, n=2. (C) Representative example of immunofluorescence staining of N protein (green) after SARS-CoV-2 infection of Calu-3 at MOI 0.4 TCID50VERO/cell, at time points shown. Nuclei (DAPI, blue), cell mask (red). Scale bar represents 50µM. (D) Quantification of N staining in cells in (C) by immunofluorescence . (E) Infectious virus released from cells in (A) determined by TCID50 on Vero.E6 cells. Fold induction of (F) interferons (IFNβ, IFNλ1 and IFNλ3) pro-inflammatory mediators (IL-6 and CCL5) each overlaid with SARS-CoV-2 E (qRT-PCR). Fold induction of IFN stimulated genes (CXCL10 and IFIT2) each overlaid with SARS-CoV-2 E (qRT-PCR). SARS-CoV-2 infection (MOIs 0.04 (closed symbols) and 0.0004 (open symbols) TCID50VERO/cell) in Calu-3 cells with addition of 10ng/ml IFNβ before or after infection at time points shown, measured by (I) E RNA copies all measured at 24 hpi. Dotted line indicates untreated. Mean +/- SEM, n=3, One-way ANOVA Light and dark blue * indicates significance for high and low MOIs respectively. SARS-CoV-2 infection (MOIs 0.04 (closed symbols) and 0.0004 (open symbols) TCID50VERO/cell) in Calu-3 cells with addition of 10ng/ml IFNβ before or after infection at time points shown, measured by (J) N positive cells, (K) released virus (TCID50VERO/cell) all measured at 24 hpi. Dotted line indicates untreated. Mean +/- SEM, n=3, One-way ANOVA Light and dark blue * indicates significance for high and low MOIs respectively.
LMF1 contributes to redox homeostasis in the ER.Figure 4Differential sensitivity of HEK293 cells containing and lacking LMF1 (A) to drugs that perturb cellular redox homeostasis. Cells were treated with BSO, DTT, or tunicamycin as described in materials and methods. Treated cell and untreated cells were counted, and the fraction of surviving cells was calculated. Three wells were counted and averaged for each data point, and three independent trials were carried out per condition. Significance was determined by a 2-tailed student's t-test. Differential sensitivity of cld/cld and wt/cld MEFs (B) to drugs that perturb cellular redox homeostasis. Cells were treated with BSO, DTT, or tunicamycin as described in materials and methods. Treated cell and untreated cells were counted, and the fraction of surviving cells was calculated. Three wells were counted and averaged for each data point, and three independent trials were carried out per condition. Significance was determined by a 2-tailed student's t-test.  C). The redox sensor ERroGFP-S4 shows that HEK293∆LMF1 cells have a more oxidized ER than HEK293 and HEK293+LMF1 cells both untreated and when stressed by expression of LPL-mCherry. A two-tailed student's t-test was used to calculate the significance of the difference in the ratio of oxidized/reduced GFP fluorescence intensity between cell types. Each data point is one of three spots measured per cell, 17 cells were measured per condition. Significance was determined by a 2-tailed student's t-test
Fig.Figure 9Multiple filaments per SVs are formed at the ribbon, SVs with Ri. + In. increase upon stimulation in wild-type (1). SVs are transported towards the membrane via the presynaptic density (2). After single-tethering at the AZ (3), multiple-tethering (4) occurs. After docking (5), SVs fuse (6). In OtofPga/Pga IHCs fewer SVs with connections to the ribbon and each other are found (1') compared to wild-type. After transport to the AZ-membrane (2'), single-tethered SVs are found (3'), and multiple-tethered and docked SVs accumulate (4', 5').
Figure 1A. NIH3T3 cells transfected with KV10.1*EGFP did not show primary cilia. Cells were transiently transfected, after 24 h serum was removed for additional 24 hours to induce ciliogenesis, and finally cells were stained with anti-acetylated α-tubulin. While most cells were ciliated, those showing green fluorescence were devoid of cilia. Scale bar: 10µm. B. NIH3T3 cells transfected with KV10.1 (red bars) showed markedly less cilia than control cells (empty vector, white bars). Subconfluent cultures grown in the presence of FCS were serum starved for 24 hours to induce ciliogenesis. Cilia were stained with anti acetylated α-tubulin antibody. To determine ciliary disassembly, cells were starved for 24 hours and then incubated for 4h in FCS to induce cell cycle re-entry and ciliary resorption. C. Similarly, hTERT-RPE1 cells transfected with KV10.1 also showed less cilia. Ciliogenesis and ciliary disassembly were induced as in B and cilia were stained using anti acetylated α-tubulin and quantified as in A. The Inset shows the equivalent experiment using the structurally related potassium channel KV10.2, which did not alter the frequency of expression of cilia.D. Examples of fields of view of hTERT-RPE1 cells transfected with KV10.1, serum starved for 24 hours and cilia were revealed with anti Arl13B antibody (arrows). A majority of control-transfected cells showed cilia, while KV10.1 transfected did not. Scale bar: 10µm.
In JAK2WT cells, Mpl is detected at the cell surface and colocalizes with JAK2Confocal fluorescent images of HEL cells (JAK2V617F+/+) after immuno-fluorescence labeling of endogenous Mpl (A), or after transient transfection with cDNA coding for a chimeric Mpl protein fused to mOrange2 (B).Figure 2Confocal fluorescent images of HEL cells (JAK2V617F+/+) after immuno‐fluorescence labeling of endogenous Mpl (A), or after transient transfection with cDNA coding for a chimeric Mpl protein fused to mOrange2 (B). C) Confocal fluorescence image of K562 cells (JAK2WT+/+) transiently transfected with cDNA coding for MplmOrange2. Red arrow in this image points to plasma membrane localization. D and E) Confocal fluorescent images obtained after transient expression of MplmOrange2 and immuno‐fluorescent labeling of endogenous JAK2 in K562 (D) or HEL (E) cells. Profiles of fluorescence (across individual cells and indicated by a white arrow) are showing a good colocalization of Mpl and JAK2WT in K562 cells at the plasma membrane (black arrow heads) but no colocalization have been observed between MplmOrange2 and JAK2V617F in HEL. F and G) Confocal fluorescent images obtained after transient coexpression of MplmOrange2 and either JAK2WTmCitrine (F) or JAK2V617FmCitrine (G) in K562 cells. Profiles of fluorescence (white arrows) are showing a very good colocalization of MplmOrange2 with JAK2WTmCitrine, especially at the plasma membrane (black arrow heads), but a very poor colocalization with JAK2V617FmCitrine (grey arrow heads). Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.
Figure 4A Genomic attributes of RNA associated with renatured NF-H, Tau, or Aβ. Inset shows which fractions of the peaks that derive from exons or introns have their origin in coding or non-coding transcripts, respectively. B Characterization of DNA associated with soluble, renatured Aβ or all proteins (Total), captured either by immunoprecipitation (Aβ ) or absorption to membranes (Total). C Sequence logo of the computationally identified motif (M1) in the solubilising genomic DNA of the Total samples.
KRAS alterations in human MPM from published datasets and the cancer genome atlas (TCGA) pan-cancer MPM cohort.Figure 1A KRAS and TP53 mutation frequencies in MPM from the catalogue of somatic mutations in cancer (COSMIC) stratified by histologic subtype (n = 775 patients). B Top 25 mutated genes from ten molecular studies of human MPM (n = 838 patients). C-E KRAS and TP53 alterations in the cancer genome atlas (TCGA) pan-cancer MPM dataset (n = 86 patients). Shown are clinical and molecular data plot with alteration frequencies (C) and patients reclassified as KRAS- or TP53-altered (asterisks), copy number variation data summary (D), and segments of the KRAS and TP53 loci (E).
Signatures of immune mediators in asymptomatic and symptomatic COVID-19 patients.Figure 2A. Heatmap of immune mediator levels in plasma samples of healthy controls (HC) (n=23) and COVID-19 patients who are asymptomatic (n=48), or symptomatic (n=172). First plasma sample from each SARS-CoV-2 PCR-positive patient was extracted for analyses. Each color represents the relative concentration of a particular analyte (blue=low concentration; red=high concentration). Each row represents one patient. Hierarchical clustering was performed on patients in each severity stratum using mEV software. B. Network analysis of significant immune mediators between symptomatic and asymptomatic COVID-19 patients. Interactive relationships between the cytokines or chemokines were determined by STRING (Search Tool for the Retrieval of Interacting Genes/ Proteins) analysis, with a confidence threshold of 0.5.
Figure 1STRING diagram representation of the network of proteins involved in NF-κB pathway. The Cancer Genome Atlas (TCGA RNAseq dataset) was used on the GlioVis platform (Bowman et al, 2007) to analyze the probability of survival (log-rank p-value) of 155 GBM patients, for each gene encoding for the mediators of the NF-κB pathway. Kaplan-Meier curve of the probability of survival for 155 GBM patients with low or high MALT1 RNA level, using median cut-off, based on the TCGA RNAseq dataset. Box and whisker plot of MALT1 mRNA expression in low-grade glioma (LGG, grades II and III) or in GBM (grade IV) (TCGA GBMLGG, RNAseq dataset) (D). Alternatively, MALT1 mRNA expression was plotted in non-tumor samples versus GBM samples (TCGA RNAseq dataset) (E). Each dot represents one clinical sample. Fraction of surviving cells over time in GSC#1 and GSC#9, transduced with control (shc) or bi-cistronic GFP plasmids using two different short hairpin RNA (shMALT1 sequences, seq #1 and #2). Data are plotted as the percentage of GFP-positive cells at the day of the analysis (Dx), normalized to the starting point (Day 4 post-infection, D4). EdU incorporation (green, 2 hours) was visualized by confocal imagery in GSC#1 or by FACS in GSC#9 transfected with sic or siMALT1. In GSC#1 the percentage of EdU-positive cells was quantified. Nuclei (DAPI) are shown in blue. n> 240 cells per replicate. Scale bar: 10 μm. Data are presented as the mean + s.e.m. on 3 independent experiments. FACS analysis of propidium iodide (PI) staining in GSC #1 and #9 transfected with non-silencing duplexes (sic) or MALT1 siRNA duplexes (siMALT1) and analyzed 72 hours later. Linear regression plot of in vitro limiting dilution assay (LDA) for control (shc) or shMALT1 seq#1 and seq#2 transduced GSC#9. Data are representative of n=2. Knockdown efficiency was verified at day 3 by western-blot using anti-MALT1 antibodies. GAPDH served as a loading control. Tumorspheres per field of view (fov) were manually counted in sic or siMALT1 transfected GSC#1, #4 and #9. Data are presented as the mean + s.e.m. on 3 independent experiments.
Model of neurexin bound to both neurexophilin and neuroligin in the synapse.Figure 6A hypothetical composite structure of Nrxn1 bound to both Nxph1 and Nlgn1 is shown. The Nxph1-LNS2SS2A+ complex structure was superimposed on the near full-length crystal structure of Nrxn1α (3R05) containing LNS2-LNS6 domains (Chen et al, 2011). Note that there are no coordinates for the Nrxn1α LNS1 or EGFA domains; structures shown here are models based on the EGFC and LNS6 domains of Nrxn1α (3R05). Nlgn1 from the crystal structure of Nrxn1β-Nlgn1 complex (3BIW) (Araç et al, 2007) is modeled by superimposition of that complex with the structure of Nrxn1α. Stalk regions, trans-membrane domains, and intracellular regions for Nlgn1 and Nrxn1α were modeled based on mouse protein sequences using PyMOL. For Nrxn1, the cysteine-loop was modeled based on the reported cysteine-loop sequence (Sterky et al, 2017), and glycosylation of the stalk region was modeled based on predicted O-linked glycosylation sites (Steentoft et al, 2013). Note the opposite effects on binding affinities of inserts at SS2 and SS4 for the Nrxn1 LNS2-Nxph1 and Nrxn1β-Nlgn1 complexes, respectively.
Figure 4 A. ChIP-qPCR confirmation of Rif1 and Rif1-∆C594 association with replication origins. ChIP was performed using cells arrested in α-factor (open bars) or HU (grey bars), and qPCR analysis performed for early origin ARS1426 (left) and late origin ARS1412 (right). Values shown are 'normalised ChIP efficiencies' obtained by subtracting the value obtained at a control locus (see Materials and Methods). Bars indicate the averages of two biological replicates, with values from each replicate shown by open circles   B. Numbers of early and late origins associated with Rif1 and Rif1-∆C594 peaks. Plot showing numbers of early and late origins bound by Rif1 and Rif1-∆C594 in G1 and HU-blocked cells, based on peak-calling results   C. Replication timing programme is intact in rif1-∆C594 cells. Replication of selected origins at an HU block analysed by BrdU incorporation. Cells were synchronised by α-factor and released into the medium containing 0.2 M HU and 1.13 mM BrdU. Plots show the percentage of total ARS607, ARS422.5, or ARS1412 DNA pulled down by IP with anti-BrdU, calculated from two biological replicate samples. Bars indicate the average of two biological replicates and open circles the values from each replicate. Insets in ARS422,5 and ARS1412 panels show the same data with Y-axis scales adjusted to 0 - 0.03%   D. Peaks of Rif1 become broader in HU-arrested cells. Box and whisker plot shows peak widths of full length Rif1 at early and late origins in G1 and HU-blocked cells. Analysis was performed on those origins detected by peak calling as associated with a Rif1 peak. Boxes show the range of 25th to 75th percentiles, with horizontal lines within the boxes representing 50th percentiles. Whiskers represent 95% confidence intervals. Outliers are presented as open circles. Numbers of origins analysed are: 46 Early origins in G1, 56 Late origins in G1, 85 Early origins in HU, and 24 Late origins in HU