EMBO/sd-character-level-ner huggingface dataset:
GBS pigment induces membrane permeabilization and K+ efflux independently of the NLRP3 inflammasome.Figure 5A, B THP-1macrophages proficient for NLRP3 (transfected with scrambled control, A) or deficient for NLRP3 (shNLRP3, B) were treated with 1 μM pigment or ΔcylE extract for 20 min, and propidium iodide (PI) was added during the final 10 min. PI uptake was measured by flow cytometry, and data shown are representative of two independent experiments. C, D Intracellular potassium concentration was measured by ICP-AES. THP-1macrophages transfected with the scrambled control (C) or shNLRP3 (D) were treated with GBSpigment (1 μM) or an equivalent amount of the ΔcylE extract. At various time points, cells were lysed and intracellular [K+] was measured relative to untreated cells (see bars and left y-axis), and percent cell death was quantified by alamar blue (see squares, dotted connecting lines and right y-axis). Both NLRP3-proficient and NLRP3-deficient macrophages initially lose intracellular K+ due to GBSpigment (compare t = 0 min to t = 30 min), but the NLRP3-deficient cells (shNLRP3) are able to recover, while the scrambled control do not, demonstrating that initial K+ loss occurs independently of NLRP3. Data are average of three independent experiments performed with independent pigment preparations in triplicate and were analyzed using Dunnett's multiple comparison test following ANOVA; all data were compared to control at t = 0, error bars ± SEM. (C) n = 3, ***P = 0.0002, *P = 0.019, **P = 0.0032 for 120 min, **P = 0.0072 for 180 min. (D) n = 3, **P = 0.0043, *P = 0.031. (E) WT THP-1macrophages were incubated with pigment in media containing either 5 mM or 50 mM potassium chloride, and cytotoxicity was measured by alamar blue assay. The addition of potassium chloride is able to protect the macrophages from cytolysis, demonstrating that K+ efflux is essential for this process. Data shown are three independent experiments performed in triplicate and were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM (n = 3, ****P < 0.0001, **P = 0.0036).
Red1 foci are dynamic during meiosis.Figure 7(A) Red1 signals disappeared at early in meiosis and then reappear in spores. Representative deconvolved images of a strain expressing both Red1‐tdTomato and CFP‐Mmi1 during meiosis are shown. DNA was stained with DAPI. Bars, 2 μm. (B) The protein level of Red1 does not change during meiosis. Cell lysates were prepared from a myc‐tagged Red1‐expressing strain in a vegetatively growing state or arrested at meiotic metaphase I and then analysed by western blotting. Cdc2 levels were also examined as a loading control. (C) Cell conjugation was not required for the loss of Red1 foci. fus1Δ cells expressing both Red1‐tdTomato and CFP‐Mmi1 were cultured with (+N) or without (−N) a nitrogen source at 26°C. Representative images are shown. The shapes of cells are indicated with white dotted lines. Bars, 2 μm. (D) Pheromone signaling but not nitrogen starvation triggers the disassembly of Red1 foci. h− strains carrying Red1‐tdTomato, Red1‐tdTomato/matPc, or Red1‐tdTomato/matPc/mei2Δ were grown in the presence or absence of nitrogen for 12 h at 26°C. The white dotted lines indicate cell shape. Bars, 2 μm.
GRASP55 controls autophagy initiation.Figure 5(A, B) Effect of GRASP55 on autophagy induction by measuring LC3‐II. BMM cells were transfected with GRASP55 siRNAs or scramble (Scr) control. At 72 h post transfection, cells were induced for autophagy, treated or not with Bafilomycin A1 (Baf) to inhibit autophagic degradation and LC3‐II/actin ratios determined by immunoblotting (A) followed by densitometry (B). Data represent mean values±s.d. (n⩾3); *P0.05. (C, D) RAW 264.7 was transfected with GRASP55 siRNAs or scramble (Scr) siRNA control. Following 48 h of siRNA treatment, cells were transfected with RFP-GFP-LC3 plasmid (GFP is sensitive to acidification, whereas RFP is not), after 24 h induced for autophagy in EBSS for 1 h and autophagic induction and flux quantified (graph in D) by determining the number of early autophagic organelles (GFP+RFP+ puncta) and autolysosomal organelles (GFP−RFP+ puncta) per cell as illustrated in fluorescent images (yellow arrows, GFP+RFP+; red arrows, GFP−RFP+). Total, yellow+red puncta per cell. Data represent mean values±s.d. (n⩾3); *P0.05. Figure source data can be found in Supplementary data.
Genetic ablation of iNOS prevents LPS-driven deregulation of the TCA cycle and energy production.Figure 2Scores scatter plot of partial least square discriminant analysis (PLS-DA) model of metabolites from WT and iNOS KO mice treated with or without LPS. Metabolomic data were range-scaled and mean-centered. Pathway Analysis using MetaboAnalyst 4.0 Software comparing significantly altered pathways from WT saline to LPS as well as iNOS KO saline to LPS. Pathways are ranked by their significance and filtered based on a Pathway Impact Score >0.1. Metabolomic data were range-scaled and mean-centered. P-values were obtained using GlobalTest and the -log(p-value) corresponding to a p-value of 0.05 is indicated by the red dashed line. (left) Western blot analysis of pThr172-AMPK (pAMPK) and total AMPK (AMPK) in quadriceps muscle. (right) Quantification of the pAMPK to AMPK ratio. Ratios are expressed relative to the saline treated controls. Schematic of Glycolysis and the TCA cycle. White boxes denote detected metabolites. Grey boxes denote undetected metabolites. Dashed arrows indicate presence of multiple reactions between metabolites. Relative concentrations of metabolites involved in glycolysis and TCA cycle. Relative concentration of arginine. Schematic of acylcarnitine metabolism. Grey circles denote key enzymes involved in metabolic processes highlighted. Relative estimated activity of CPT1, CPT2, LCADH, and β-oxidation.
Characterisation of the two lamellocyte clusters.Figure 6A) Scatter plot comparing the transcriptome of the two lamellocyte clusters, deduced from the WI dataset. The x-axis is the average expression levels of the genes and the y-axis the log2 fold change (LM-1/LM-2). The genes significantly enriched in the LM-1 cluster are highlighted in red (p<10-6), those significantly enriched in the LM-2 cluster in orange (p<10-6). B) GO term enrichment analysis on the genes enriched in lamellocytes compared to all other clusters (top panel) and on the genes specific to LM-1 (middle panel) or LM-2 (lower panel). The bars represent the fold enrichment and the color gradient indicates the p-value of the GO term enrichment C) Expression levels (gradient of color) and the percentage of cells (size of the dots) for the top 10 markers of lamellocytes (in black), of markers specific to LM-1 compared to LM-2 (in red) and specific to LM-2 compared to LM-1 (in orange). Note that most markers enriched in LM-1 compared to LM-2 are exclusively expressed in LM-1 while most markers enriched in LM-2 compared to LM-1 are also expressed in the plasmatocyte and crystal cell clusters. D) Hemocytes from WL OregonR infested by wasp. The immunolabelling was done with antibodies targeting the new lamellocyte markers identified in this study Rhea (in gray, top panels) and Shot (in gray, lower panels). Phalloidin-FITC (in green) labels the actin filament particularly abundant in lamellocytes (Tokusumi et al., 2009) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI, FITC and the lamellocyte markers, the middle panels show the FITC alone and the right panels the lamellocyte markers alone. The scale bars represent 20µm.
Figure 4A. Angptl2 mRNA levels in small intestine (proximal, mid and distal), cecum and colon (proximal and distal) of wild-type mice based on qRT-PCR analysis. n=5. B. ANGPTL2 IHC in colon from wild-type and Angptl2-/- mice. The Angptl2-/- colon image serves as a negative control. Scale bar = 50 μm. C. mRNA levels of indicated genes in wild-type (n=4) mice based on qRT-PCR analysis. D. Representative IF images of distal colon from wild-type mice as assessed with anti-ANGPTL2 (red) and anti-α-SMA (green). Nuclei are counterstained with DAPI (blue). Scale bar = 100 μm.
Increased glycolysis, reduced drug uptake or mutations in the drug target do not explain IMT1-induced resistance A ECAR measured by Seahorse extracellular flux analysis in DMSO and IMT1-treated RKO and IMT1-resistant cells.Figure 5A ECAR measured by Seahorse extracellular flux analysis in DMSO and IMT1-treated RKO and IMT1-resistant cells. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test for multiple comparisons. RKO DMSO vs RKO + IMT1: **p=0,0019; RKO + IMT1 vs resistant + IMT: *p=0,0214; RKO DMSO vs resistant DMSO: non-significant. B Bioenergetic profiles measured with Seahorse flux analyzer, showing the contribution of OCR and ECAR to cellular bioenergetics in RKO and IMT1-resistant RKO cells treated with and without IMT1. Mean of n=3 independent experiments ± SEM. C Dose-response viability curves during treatment with serial dilution of IMT1 for one-week in RKO and IMT1-resistant RKO cells. Data are expressed as the mean ± SD of n=3 experiments. D, E IMT1 extracellular (D) and extracellular (E) concentrations measured in the medium at 0, 2 and 24 hours of IMT1 treatment in RKO and IMT1-resistant RKO cells. Mean values ± SEM of n=3 experiments. F Schematic representation of the IMT1 binding region on the human POLRMT sequence, adapted from Previously identified mutations conferring resistance to IMT1 are reported in red Representative results of n=3 independent sequencing experiments of the POLRMT gene in RKO and IMT1-resistant RKO cells at the region of interest.
Figure 1Sites that bound either HIF-1α (A & B) or HIF-2α (C&D) in both replicates were identified by the MACS peak caller and ordered on the y-axis according to HIF-1β signal intensity. Heatmaps show HIF ChIP-seq signal (read counts per million mapped reads, CPM; expressed as colour intensity, averaged across two independent ChIP-seq experiments) at HIF binding sites and across the flanking ±5kb regions (x-axis). (A) HIF-1β signal intensity and (B) HIF-1α signal intensity at HIF-1α binding sites and (C) HIF-1β signal intensity and (D) HIF-2α signal intensity at HIF-2α binding sites. HIF-1β signal intensity above local background levels is observed at all HIF-1α and HIF-2α binding sites. (E & F) Line plots showing average (n = 2, independent ChIP-seq experiments) HIF-1β signal intensity (solid blue lines) within the MACS defined (E) HIF-1α and (F) HIF-2α binding sites compared to the average background HIF-1β binding signal (dashed black line) at non-HIF-α-binding accessible sites (defined by FAIRE-seq). Sites are ranked on the x-axis according to HIF-1β signal. HIF-1β signal intensity at both HIF-1α and HIF-2α binding sites was consistently above genome-wide background levels. (G) HIF-1β signal intensity was plotted against total HIF-α (HIF-1α + HIF-2α) signal intensity for all sites that bound one or more HIF subunits. Strong correlation was observed between HIF-α and HIF-1β signal intensities. (H) The ratio of total HIF-α signal to HIF-1β signal (x-axis) was determined for each site and plotted as a frequency distribution (y-axis) showing a tight unimodal distribution. (I) The non-reproducibility of the total HIF-α/HIF-1β ratio was assessed by plotting the ratio in replicate 1 versus that in replicate 2
Figure 4(A) Outer membrane damage (mCherry intensity) and inner membrane damage (% Sytox positive) of convertase-labeled perimCherry/cytoGFP E. coli cells incubated with a concentration range of C5 and fixed concentrations of C6-C9. Data information: (A-C) Data represent mean ± SD of 3 independent experiments. (B) Inner membrane damage of perimCherry/cytoGFP E. coli exposed to a concentration range of ΔC5 serum and, after washing, to C5-C9. As controls, bacteria were incubated with heat inactivated ΔC5 serum or 5 µM compstatin was added to the ΔC5 serum to block C3b deposition. Data information: (A-C) Data represent mean ± SD of 3 independent experiments. (C) Inner membrane damage of three different convertase-labeled bacteria exposed to buffer (Conv) or C5-C9 (Conv-MAC). Data information: (A-C) Data represent mean ± SD of 3 independent experiments. (C) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05 or ** p≤0.01. (D) Confocal microscopy images of convertase-labeled perimCherry/cytoGFP E. coli exposed to buffer (Conv) or C5-C9 (Conv-MAC). Unlabeled bacteria exposed to 1% serum served as control. Green = GFP, Red = To-pro-3 DNA dye. Scale bars = 3 µm.
Figure 5Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (A) EVs isolated by UC of medium from HeLa cells cultured in glutamine-replete (2.00 mM) and -depleted (0.02 mM) conditions for 24 h (see Appendix Fig S7A'' for analysis of SEC-isolated EVs). Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (B) EVs isolated by size-exclusion chromatography (SEC; fractions two to seven) from LNCaP cells cultured in glutamine-replete (2.00 mM) and -depleted (0.02 mM) conditions for 24 Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (C) EVs isolated by SEC from LNCaP cells cultured in the presence or absence of 120 nM Torin 1 for 24 h. Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (D) EVs isolated by UC from HCT116 cells cultured for 24 h following three days of raptor or non-targeting (NT) shRNA knockdown. Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (E) EVs isolated by SEC from HCT116 cells cultured in the presence or absence of 3 µM Akt inhibitor AZD5363 for 24 h. Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. (F) EVs isolated by SEC from HCT116 cells cultured under glutamine-replete conditions in the presence or absence of 10 µM KRAS inhibitor BI-2852 (or control compound Bl2853) for 24 h. (G) Table summarising the EV protein analyses in Figs 4 and 5 (normalised to cell lysate protein levels). Data analysed by the Kruskal-Wallis test: *P < 0.05. Significantly decreased levels are in blue and increased levels are in red.
Model for the roles of Rubicon and UVRAG in the maturation of autophagosomes in HCV-infected cells.Fig 8Model for the roles of Rubicon and UVRAG in the maturation of autophagosomes in HCV-infected cells.In the normal autophagic pathway, UVRAG, in complex with Beclin-1, p150 and Vps34, facilitates the fusion between autophagosomes and lysosomes to form autolysosomes. The induction of Rubicon by HCV in the early stage of infection inhibits the UVRAG activity and the fusion between autophagosomes and lysosomes. This leads to the accumulation of autophagosomes, which enhance HCV RNA replication. The induction of UVRAG in the late stage of HCV infection overcomes the inhibitory effect of Rubicon and results in the maturation of autophagosomes. In the model illustrated, the effect of HCV on the initiation of autophagy is not addressed.
Figure 1(A) Following transfection of given siRNAs and synchronization in G2-phase, Hek293 cells were DRB- or mock-treated and then subjected to IFM using the indicated antibodies. Lower panels display a 3-fold magnification of the boxed area shown above. Scales bars correspond to 5 and 1 μm, respectively  (B) Hek293 cells were thymidine-arrested for 20h, mock- or OHT-treated to induce DSBs by nuclear accumulation of ER-AsiSI and then subjected to ChIP-multiplex PCR  (C) ChIP samples from (B) were analysed by qPCR. Shown are averages (bars) of 3 independent experiments (dots) Separase interacts with γH2AX in DSB-containing G2 but not G1 cells. Transgenic Hek293 cells treated with Dox to induce expression of Myc-separase-WT and with OHT to induce nuclear accumulation of ER-AsiSI and infliction of DSBs were synchronized in G1- or G2-phase and analysed by IP-Western Separase interacts with γH2AX in DSB-containing G2 but not G1 cells. Transgenic Hek293 cells treated with Dox to induce expression of Myc-separase-WT and with OHT to induce nuclear accumulation of ER-AsiSI and infliction of DSBs were synchronized in G1- or G2-phase and analysed b IFM for γH2AX- and Myc-separase-positive foc The quantification of the IFM in (E) shows averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each
Tea2 and Klp5/Klp6/Mcp1 antagonistically control microtubule stability.Figure 2(A) Log phase cells expressing fluorescently-tagged MTs were imaged and the levels of either fluorescently-tagged Klp5/Klp6 or Tea2 at the plus ends of growing MTs determined. Measurements (klp5-mNG klp6-mNG, n=305; tea2-GFP, n=359) were plotted against microtubule (MT) length and third order polynomial curves fitted to the data (B) Kymographs showing fluorescently-tagged MTs co-imaged with fluorescently-tagged Tea2 kinesin in control cells (top panels) or in the absence of Klp6 (middle panels) or Mcp1 (bottom panels). Dashed yellow line indicates the cell end  (C) Intensity of Tea2 at plus ends quantitated from multiple kymographs of the type in (B) for control (n=16), ∆klp6 (n=13) or ∆mcp1 (n=14) cells (D) Kymographs showing fluorescently-tagged MTs co-imaged with fluorescently-tagged Klp5/Klp6 in control (top panels, repeated from 1C) or ∆tea2 cells (bottom panels). Dashed yellow line indicates the cell end (E) Cells expressing fluorescently-tagged tubulin were imaged every 5 seconds and the dwell time of ~100 individual iMTs for each condition recorded within the final 1.1 µm of the cell. Red bars signify the mean  (F) The strains in (E) were analysed to determine the position of 20 MT shrinkage events relative to the cell end, which are represented both graphically and as a box plot
Figure 8A. Representative Per2::Luciferase trace of Cry1-/- SCN slice at baseline (black) and after AAV-mediated (arrows) Cry1 complementation in Prok2+ cells (red) or ProkR2+ cells (yellow). B. Corresponding peak-to-peak period plots for baseline (black), Cry1-expressed in Prok2+ (red), and then in ProkR2+ cells (yellow). Individual slices are depicted as grey traces, filled points are their mean±S.E.M. C. Group data (n=5) across three Per2::Luciferase cycles as shown in B. (p=0.006; baseline vs. Prok2.Cre *p=0.04, baseline vs. Prok2.Cre and ProkR2.Cre *p=0.02, Prok2.Cre vs. Prok2.Cre and ProkR2.Cre ns. p=0.95). D. As in A but with order of transfection reversed; green =Cry1 only in ProkR2+ cells (baseline vs. ProkR2.Cre *p=0.04). As in B and C but with transfection order reversed (green =Cry1 expression in ProkR2+ cells only, n=6) (p=0.006; baseline vs. ProkR2.Cre *p=0.03, baseline vs. ProkR2.Cre and Prok2.Cre **p=0.009, ProkR2.Cre vs. ProkR2.Cre and Prok2.Cre ns. p=0.1). F. As in B and C but with transfection order reversed (green =Cry1 expression in ProkR2+ cells only, n=6) (p=0.006; baseline vs. ProkR2.Cre *p=0.03, baseline vs. ProkR2.Cre and Prok2.Cre **p=0.009, ProkR2.Cre vs. ProkR2.Cre and Prok2.Cre ns. p=0.1). G. The final circadian periods of Cry1-/- SCN slice double-transduced in complementary reverse orders did not differ between each other, p=0.7.
Figure 1MPRNA scheme and hnRNPK IP Western blot. The blot image is a section of the image shown in Figure EV6A. Volcano plot of the RIP-seq data. Each point represents a gene. Red points correspond to the 146 genes with log2(IP/Input)≥0.5 and adjusted P<0.1. P-values computed using Wald test as implemented in DESeq2. hnRNPK IP enrichment ratios for genes with the indicated number of exonic hnRNPK eCLIP clusters in ENCODE data from HepG2 cells (average of the two ENCODE replicates). Enrichment ratios are log2-transformed fold changes computed by DESeq2 based on four replicates. The box plots show the interquartile range (IQR) and the line indicates the median value. Whisker ends extend to 1.5 times the length of the IQR (unless the data range is smaller, in which case the whisker ends extend to the minimum or maximum value), points indicate outliers. Spearman's correlation R and P-value are indicated. Nuc/Cyto ratios in MCF-7 cells (ENCODE data) for genes bound by hnRNPK (from (B)) and other genes. Enrichment ratios and boxplots are as in C, P-value computed using two-sided Wilcoxon rank-sum test. Change in the Nuc/Cyto ratios (log2) between sihnRNPK- and siNT-treated cells, for the genes bound by hnRNPK (from (B)) and other genes. Enrichment ratios, boxplots, and statistical test are as in D. As in (E), for changes in gene expression, computed by DESeq2. Correspondence between Nuc/Cyto ratios induced by each tile in NucLibB and its hnRNPK IP/Input ratio. Coloring indicates local point density. Spearman's correlation R and P-value are indicated. For each sequence in NucLibB containing repeats of the indicated 6-mer or 10-mer, the Nuc/Cyto (green) and hnRNPK IP/Input (orange) ratios are shown. Vertical lines show the corresponding values for the Jpx#9 and Pvt1#22 tiles.
enhances inflammasome activation to benefit ZIKV infection.Figure 2(A) Relative qRT-PCR analysis of ZIKV RNA in THP-1 cells left untreated or treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs), followed by ZIKV infection (MOI=1) for the indicated time points. (B) Relative qRT-PCR analysis of ZIKV RNA in wild type (WT) or NLRP3 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points. (C) Immunoblot analysis of extracts of WT or Nlrp3-/- BMDMs by the indicated antibodies. (D) Relative qRT-PCR analysis of ZIKV RNA in WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points. (E) Plaque titration of ZIKV in supernatants of WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points. (F) Immunoblot analysis of extracts of WT or caspase-1 KO THP-1 cells by the indicated antibodies. (G) Relative qRT-PCR analysis of ZIKV RNA in WT or caspase-1 KO THP-1 cells infected with ZIKV (MOI=1) for the indicated time points. (H) Relative qRT-PCR analysis of ZIKV RNA in NS-1-inducible THP-1 cells treated with or without doxycycline (Dox), then left untreated or treated with Ac-YVAD-cmk (20 μΜ) for 3 hrs followed by ZIKV infection (MOI=1) for the indicated time points. (I) Relative qRT-PCR analysis of NLRP3 knockdown efficiency in PBMCs from two donors. (J) Relative qRT-PCR analysis of ZIKV RNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV (MOI=1) for 36 hrs. (K) qRT-PCR analysis of ZIKV RNA in PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.
Figure 3iTRAQ proteomics analysis (Appendix Fig. S2) resulted in 2,100 proteins consistently quantified at every point across the IDC. The heat map shows hierarchical clustering analysis of fold-changes (log2-transformed) in the levels of the proteins relative to an arbitrary average across the IDC. The 100 most up-regulated proteins at each time point were subjected to pathway enrichment analysis based on GO and MPM pathways annotated for P. falciparum 3D7. The significantly (P < 0.05) enriched pathways among the up-regulated proteins at each time point are summarized in tables for each time point