EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) qRT-PCR analysis of defense-related genes in 10-day-old seedlings exposed to 0.5 µM Pep2 for 10 h. (*, p < 0.05 in two-tailed tests compared to the corresponding WT values. Relative Ct values of two independent experiments with three biological replicates each were combined for statistical analysis.) (B) Root length (averages ± SD, n ≥ 15) of Arabidopsis seedlings exposed to 100 nM Pep2. (*, p < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of WT plants. Two independent experiments were combined for statistical analysis.) (C) Fresh weight (averages ± SD, n ≥ 25) of 2-week-old plants grown on soil. (*, p < 0.05 in two-tailed tests compared to the corresponding values of WT plants. ■, p < 0.05 in two-tailed tests. Two independent experiments were combined for statistical analysis.) (D) Immunoblot analysis for BAK1 in 10-day-old seedlings with or without 0.5 M Pep1 for 24 h. The numbers below the upper panel represent relative intensities of the BAK1 band normalized to the backgrounds in the Ponceau S-stained loading controls, with the value in non-elicited WT plants set as 1.0.
Figure 4A) srd-1 mutant males were not attracted to the sex pheromone but were attracted to the positive-control chemoattractants, 1:1000 diacetyl and 10 mg/mL pyrazine. This demonstrated that SRD-1 is required and specific for sex pheromone perception. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown. B) Summary of srd-1 expression pattern. The transcriptional reporter Psrd-1::gfp was detected in both male and hermaphrodite ASI neurons in the head region, and only in male AWA and ADF neurons. C) SRD-1::GFP fusion construct revealed predominant SRD-1 localization at the cilia of AWAs in C. elegans males. Scale bar = 5 μm. D) Examination of srd-1 transcriptional reporter revealed that srd-1 is expressed in AWA neurons in males but not hermaphrodites. A transcriptional reporter for odr-7 indicated the locations of AWAs (arrowheads). Scale bar = 50 μm. E) Schematic diagram of reprogramming of chemotaxis in C. elegans. Rescue-experiment results demonstrated the necessity of SRD-1 in sex pheromone perception; ectopic expression of SRD-1 in the chemorepulsion-responsible neuron, AWB, revealed that this SRD-1 expression is sufficient for altering sex pheromone preference in C. elegans. F) AWA-specific rescue of srd-1 restored sex pheromone perception ability in both males and hermaphrodites. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown. G) Ectopic expression of srd-1 in AWB neurons elicited a distinct repulsive behavior toward the sex pheromone in both males and hermaphrodites. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.
Tollip translocates to a LAMP1 compartment during mitochondrial stress.Figure 6(A) Quantitation of Tollip vesicular clusters in SH-SY5Ys treated with 5 μM antimycin A/10 μM oligomycin B (AO) for 2 hours. The percentage of cells with vesicular clusters were quantified from 7-23 cells for each condition from 4 independent experiments. (B) Quantitation of the prevalence of Tollip clusters in HEK293 WT or Tom1 KO cells treated with AO for 2 hours. The percentage of cells with vesicular clusters were quantified from an average of 50 cells for each condition from 5 independent experiments. (C) Endogenous Tollip and LAMP1 colocalise in SH-SY5Y cells treated with AO for 2 hours. Antibodies specific to LAMP1 (green) and Tollip (red) were used. (D-E) SH-SY5Y cells were transfected with either wild-type GFP-Tollip (WT), GFP-Tollip lacking its N-terminus (∆Nterm), or GFP-Tollip containing a CUE domain mutation (CUEmut) then treated with AO for 2 hours. Cells were fixed and immunostained with antibodies specific to GFP (green) and LAMP1 (red). (F) The extent of colocalisation between GFP-Tollip and LAMP1 from untreated and AO treated SH-SY5Y cells were quantitated from immunofluorescence images and represented as a Pearson's correlation (2-3 cells per experiment, from 2 independent experiments). (G-I) The extent of colocalisation between GFP-Tollip and LAMP1 in Tom1 KO (G), ATG5 KO (H), and VPS35 KO (I) compared to wild-type (WT) HeLa cells was quantified from immunofluorescence images immunostained for GFP and LAMP1 and represented as a Pearson's correlation (2-3 cells per experiment, from 3 independent experiments). Western blot analysis of lysates harvested from wild-type (WT) alongside either Tom1 KO (G) or VPS35 KO (I) Hela cells was performed using antibodies specific to the indicated proteins.
Figure 1A. Schematic representation of the experimental protocol. Abbreviations: LCM, laser capture microdissection. B, C. RT-PCR performed on RNA extracted from axons collected by LCM (Ax) or from eyes. (B) β-Actin mRNA is present both in eyes and axons, while MAP2 and H4 are present only in the eye sample, suggesting the absence of contamination from cell bodies or other cells in LCM axonal samples (Bellon et al, 2017). Abbreviations: Ax, axonal sample; Eye, stage 37/38 eye; LCM, laser capture microdissection; -, PCR no template control; NT, RT no template control. D. Schematic of molecular beacon (MB), pre-miR-181a-1 and their hybridization complex. Abbreviations: MB, molecular beacon. E. Schematic of thermal denaturation profile of the MB. Abbreviations: MB, molecular beacon. F. Thermal denaturation profiles of MB, in the absence (solid line) and presence (dashed line) of increasing target concentration. Each melting curve represents the average of three separate replicates. Yellow boxes indicate the range of working temperatures for ex vivo trafficking experiments. Abbreviations: MB, molecular beacon; Tm, MB melting temperature. G. Schematic representation of the experimental protocol. Concentrations used: 5µM MB; 250µM co-MO; 250µM pri-miR-MO. Abbreviations: MB, molecular beacon; co-MO, control morpholino; pri-miR-MO, morpholino blocking pre-miR-181a-1 processing by targeting the Drosha cleavage site. H. Quantification of the expression levels of pre-mir-181a-1 using the 2-ΔCt method and U6 as normalizer from small total RNA fraction (<~150 nt). Each data point corresponds to one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant. I. Total number of MB puncta normalized to axon length (µm). Each data point corresponds to one axon. Total number of puncta and axons analyzed: 928 puncta, 61 axons (WT), 226 puncta, 15 axons (co-MO), 208 puncta, 35 axons (MO). n=4 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino; pri-miR-MO, morpholino blocking pre-miR-181a-1 processing by targeting the Drosha cleavage site. J. Representative axons. MB puncta are indicated (white arrows). Dashed white lines delineate axons. Abbreviations: MB, molecular beacon. Scale bars: 5µm. K. Schematic representation of the experimental protocol. Concentrations used: 5µM MB; 200-250ng/µL cy3-pre-miR-181a-1. Abbreviations: MB, molecular beacon. L. Schematic representation of cy5-labeled pre-miR-181a-1. M. Frequency (in percentage) of puncta colocalization between MB and cy5-pre-miR-181a-1 (#MB+/pre-miR+). Each data point corresponds to one axon. Total number of puncta and axons analyzed: 354 puncta, 32 axons (MB), 337 puncta, 32 axons (cy5-pre-miR-181a-1). n=5 independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon. N. Representative image of RGC axons. White, red and blue arrows indicate, respectively, co-localized, single MB, and single pre-miR-181a-1 puncta. Scale bars: 5µm.
Figure 6(A) Immunoblot analysis of fibronectin, pSmad2/3, total Smad2/3, and GAPDH (loading control) in cell lysates from MCF10A-pIND20-HPN cells without hepsin overexpression. The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels, compared to siCtrl (N = 3 biological repeats). (B) Immunoblot analysis of cell lysates (pSmad2/3, total Smad2/3 and vinculin as loading control) and concentrated cell culture supernatant (anti-V5 for TGFβ) from MCF10A-pIND20-HPN pL6-TGFβ1-V5 cells without hepsin overexpression. Ponceau staining is shown as a loading control for the western blot with concentrated culture supernatant. The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels, compared to siCtrl (N = 3 biological repeats). (C) TGFβ Luciferase reporter assay of control or fibronectin silenced MCF10A-pIND20-HPN pL6-TGFβ1 (SRE) TGFβ1 reporter cells with (+DOX) or without (-DOX) hepsin overexpression (N= 7 biological repeats; Y-axis shows fold change in Relative Light Units (RLU)). (D) Immunoblot analysis of cell lysates (fibronectin and β-tubulin (loading control)) and concentrated cell culture supernatant (TGFβ) from Hs578T cells with knockdown of fibronectin. Ponceau staining is shown as the loading control for the western blot with concentrated culture supernatant. A representative blot from 3 biological repeats is shown. (E) Immunoblot analysis of fibronectin, pSmad2/3, total Smad2/3, hepsin, and GAPDH (loading control) in cell lysates from MCF10A-pIND20-HPN cells with (DOX+) and without (DOX-) hepsin overexpression, and with or without fibronectin silencing (siFN1). The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels (N = 3 biological repeats). (F) TGFβ Luciferase reporter assay of control or fibronectin silenced MCF10A-pIND20-HPN pL6-TGFβ1 (SRE) TGFβ1 reporter cells, with (DOX+) and without (DOX-) hepsin overexpression. (N=6 biological repeats; Y-axis shows fold change in Relative Light Units (RLU)). (G) Model figure depicting how hepsin regulates TGFβ signaling. Under Wt conditions, hepsin promotes degradation of fibronectin, which releases latent TGFβ from ECM stores, thus resulting in the induction of TGFβ signaling. In hepsin knockout mammary glands, TGFβ signaling is compromised as latent TGFβ cannot be released from ECM stores
Figure 4A, Heatmap representation of the 466 LC-MS features detected as rhythmic (JTK-Cycle; P<0.05, FDR=14.5%). B, Individual traces of rhythmic features from cluster 1 and 2 from A. C, Distribution of phases from A D, Metabolite Set Enrichment Analysis of the 54 identified rhythmic metabolites E, Targeted LC-MS analysis of metabolites from glycolysis, pentose phosphate pathway (PPP) and tricarboxylic acid (TCA) cycle. The colour of each node represents the p-value for daily rhythmicity (JTK Cycle). See Appendix Table S2 for list of abbreviations F, Selected temporal profiles of metabolites are shown together with their associated p-value G, Two examples of protein-metabolite pairs are shown with their respective Pearson correlation coefficients H, Heatmap showing the Pearson's correlation between each profile from targeted metabolomics (carbon metabolism) and proteomics data
iNKT cell activation by antigen presenting cells undergoing UPR A-B: Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with (A) a murine macrophage cell line (J774.Figure 1Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with (A) a murine macrophage cell line (J774.2) treated either with thapsigargin (black bars),tunicamycin (red bars) or DMSO (white bars). Graphs show mean ± SEM from n=3biological replicates. ****P < 0.0001 (Unpaired t test). Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with a human macrophage cell line (U937), treated either with thapsigargin (black bars),tunicamycin (red bars) or DMSO (white bars). Graphs show mean ± SEM from n=3biological replicates. ****P < 0.0001 (Unpaired t test). Heat map represents cytokine secretion by in vitro expanded (C) murine splenic iNKT cells co-cultured either with thapsigargin- or tunicamycin- or DMSO-treated murine BMDMs. The heat map shows the average result of two pooled biological replicates. Heat map represents cytokine secretion by in vitro expanded human peripheral blood iNKT cells co-cultured either with thapsigargin- or tunicamycin- or DMSO-treated murine BMDMs. The heat map shows the average result of two pooled biological replicates. Wild type C57BL/6 mice were injected intravenously with thapsigargin-loaded PLGA nanoparticles or vehicle, and 12 h later, hepatic iNKT cells were analysed by flow cytometry. Histograms represent expression of intracellular IL-4 and IFN-γ levels in hepatic iNKT cells from mice injected with thapsigargin-loaded PLGA nanoparticles (red histogram) or vehicle-treated (grey histogram) mice. Dot plots represents MFI for intracellular IL-4 and IFN-γ expression in hepatic iNKT cells from thapsigargin-loaded PLGA nanoparticles (red dots) or vehicle-treated (black dots) mice. Data plots show mean ± SEM from n = 5 mice per group. **P≤ 0.001 (Mann-Whitney U test).
Figure 3(A) Graphical summary of the murine sepsis model and experimental design. (B Pro- anti-inflammatory cytokines were analyzed in EDTA-plasma from peritoneal contamination and infection (PCI) and sham animals. #p<0.05 against sham, *p<0.05 as indicated; Kruskal-Wallis ANOVA with controlled false-discovery rates (Benjamini-Hochberg procedure). C) anti-inflammatory cytokines were analyzed in EDTA-plasma from peritoneal contamination and infection (PCI) and sham animals. #p<0.05 against sham, *p<0.05 as indicated; Kruskal-Wallis ANOVA with controlled false-discovery rates (Benjamini-Hochberg procedure). (D,E) Colony-forming units (CFUs) were analyzed from (D) abdominal lavage and (E) liver homogenates. Significance maps depict p-values from a Kruskal-Wallis ANOVA with controlled false-discovery rates applying the Benjamini-Hochberg procedure. (F,G) Concentration of (F) neutrophils and (G) monocytes in 2 mL peritoneal lavage. (H) Infiltrating polymorphonuclear neutrophils (PMN) were assessed from histological liver sections after immune fluorescent staining. *p<0.05 against sham, #p<0.05 against 24 h; Kruskal-Wallis ANOVA with controlled false-discovery rates (Benjamini-Hochberg procedure).
Figure 5A) Structure of SARS PLpro bound to compound 3j (cyan/yellow, pdb 4ovz, (Baez-Santos et al, 2014)) superposed with SARS2 PLpro~Ub (blue/orange). The inset shows compound 3j bound near the active site. See Fig EV5A, B for further details. B) Chemical structure of rac5c. See Appendix Supplementary Methods for detail on compound synthesis and characterisation. C) In vitro inhibition (IC50) for rac5c inhibiting SARS2 PLpro. Experiments were performed using the HTS assay (Fig 4), in technical triplicate in three independent experiments. A geometric mean was used to determine IC50. D) Full-length nsp3 was expressed from a C-terminally GFP-tagged vector in HEK293T cells and treated with increasing concentrations of rac5c for 24 h. GFP is released from the C-terminus, presumably by nsp3 protease activity. Nsp3 can be detected by a SARS/SARS2 PLpro antibody (see Fig EV5E for antibody validation). Lysates were blotted for Lys48-linked polyubiquitin with a linkage specific antibody (K48). Experiments were performed in duplicate with similar results. Also see Fig EV5F, G and Source Data for uncropped blots.
Figure 4CryoEM image of an isolated ex vivo S-Ena making a U-turn comprising 19 helical turns (shown schematically in orange), i.e. corresponding to a ~10° axial tilt between consecutive helical turns. Cross-section of the molecular surface of the ex vivo S-Ena model and the ex vivo S-Ena 3D cryoEM electron potential map. Model and map reveal a 4.5 Å longitudinal spacing between Ena1B jellyroll domains (red dashed lines) as a result of the 10 Å long Ntc linker (residues 12-17). Negative stain 2D class averages of endospore-associated S-Enas show variation in pitch and axial curvature, likely as a result of strain exerted during deposition of spores on the grid surface. The observed range in pitch corresponds to a range in longitudinal spacing of 2.5 to 12 Å between helical turns (labeled red in panel B).
Figure 3STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation. (A) HeLa cells silenced for endogenous STK38 were transfected with myc-STK38(wt) expressing plasmid, myc-STK38(K118R) (STK38 kinase-dead version) plasmid or with HA-STK38(PIF) (STK38 constitutively active version) plasmid. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours, fixed and stained for myc-tag or HA-tag. Representative images are shown and scale bars are 40 µm. STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation. (B) Graphical representation of tag-STK38 variants nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; **p<0.01, ***p<0.001, ns, not significant, Mann-Whitney test). STK38 kinase activity is required and sufficient for nutrient-starvation induced autophagy. (C) HeLa cells stably expressing the GFP-LC3-RFP-LC3ΔG autophagic probe [26] were transiently transfected with siRNA targeting the 3'UTR region of endogenous STK38 (or with non-targeting siRNA (siNT)). The next day, cells were transiently transfected with the indicated STK38 mutants expressing plasmids 24 hours after plasmid transfection, cells were incubated with DMEM or EBSS for 4 hours. The GFP-LC3 (degraded upon autophagy) and RFP-LC3ΔG (non-degraded upon autophagy) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level (n=4 independent experiments, mean ± SEM; *p<0.05, ***p<0.001, ns, not significant, Mann-Whitney test). depleting STK38 prevents autophagy to take place. This effect was partially reversed by expressing the wt ORF as well as constitutively active STK38 whereas kinase-dead version failed to reproduce endogenous STK38 effect. Expression of constitutively active STK38 is sufficient to induce a substantial change in the autophagic flux upon nutrient rich conditions STK38 kinase activity is required and sufficient for nutrient-starvation induced autophagy. (D) HeLa cells were transiently transfected with siRNA targeting the 3'UTR region of endogenous STK38 (or with non-targeting siRNA (siNT)) and then transiently transfected with the indicated STK38 mutants expressing plasmids 48h after. The next day, the cells were incubated with DMEM or EBSS for 4 hours and overall p62 level was quantified for autophagy estimation (n = 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Student's t test).
Figure 3RNA immunoprecipitation and qRT-PCR (RIP-qPCR) analysis of collagen type 1, α1 (Col1a1), collagen type 1, α2 (Col1a2), and collagen type 3, α1 (Col3a1) mRNA in CRTH2-/- and wild type (WT) fibroblasts using LARP6 antibody. *P < 0.05 vs WT (two-tailed Student's t test); n = 6 for all groups. , Effect of the CRTH2 deficiency on the stability of Col1a1, Col1a2, and Col3a1 mRNA in primary mouse fibroblasts. Fibroblasts were treated with 5 μg/mL Actinomycin D (Act D). Total RNA was extracted immediately after addition of Act D at 0, 2, 4, 6, 8, 12, and 24 h. Col1a1, Col1a2, and Col3a1 mRNA expression was quantitated by qRT-PCR. Dotted lines indicate mRNA half-lives; *P < 0.05 vs WT (two-way ANOVA); n = 6 for all groups. Effect of CRTH2 deficiency on Col1a1, Col1a2, and Col3a1 mRNA expression in primary mouse fibroblasts. GAPDH was used as housekeeping gene. *P < 0.05 vs WT (two-tailed Student's t test); n = 8 for all groups. , Effect of CRTH2 deficiency on Collagen (Col) I and III protein expression in primary mouse fibroblasts with and without TGF-β1 (10 ng/mL) treatment. Knockdown efficiency of LARP6 si-RNA (Si-LARP6) detected by qRT-PCR in primary fibroblasts. *P < 0.05 vs scramble, (two-tailed Student's t test); n = 5 for all groups. Effect of LARP6 knockdown on Col1a1, Col1a2 and Col3a1 mRNA stability in WT and CRTH2-/- fibroblasts. *P < 0.05 vs WT, (two-way ANOVA); n = 8 for all groups. , Effect of LARP6 knockdown on Col1a1, mRNA expression in WT and CRTH2-/- fibroblasts. Fibroblasts were transfected with Si-LARP6 or scramble siRNA. After 48 h transfection, RNA was prepared for qRT-PCR analysis. GAPDH was used as housekeeping gene. *P < 0.05 vs WT, #P < 0.05 vs scramble (two-way ANOVA); n = 6 for all groups. Effect of LARP6 knockdown on Col1a2, and Col3a1 mRNA expression in WT and CRTH2-/- fibroblasts. Fibroblasts were transfected with Si-LARP6 or scramble siRNA. After 48 h transfection, RNA was prepared for qRT-PCR analysis. GAPDH was used as housekeeping gene. *P < 0.05 vs WT, #P < 0.05 vs scramble (two-way ANOVA); n = 6 for all groups. Effect of LARP6 knockdown on Collagen I and III protein expression in WT and CRTH2-/- fibroblasts.
Chemical modification of canonical Wnt/β‐catenin signaling activity changes the radio‐sensitivity of cultured ISPCsCrypts of 2‐ to 3‐month‐old LGR5ki mice were cultured and γ‐irradiated with 6 Gy on day 10 of culture.Figure 7Crypts of 2‐ to 3‐month‐old LGR5ki mice were cultured and γ‐irradiated with 6 Gy on day 10 of culture. Sixteen hours before irradiation, cells were exposed to the indicated treatment (n = 4 independent experiments per group).A-J Representative FACS plots of indicated treatments are given. NIR, non‐irradiated; IR, irradiated.K-P Flow cytometry analysis of the percentage of LGR5+, LGR5hi, and LGR5lo cells of irradiated cultures (24 h after IR) compared to the percentage in non‐irradiated controls (NIR) set to 100%. (K-M) Note that inhibition of Wnt signaling by DKK1 treatment or R‐spondin reduction (50% Rspo) partially rescued the decrease in LGR5+ cells in irradiated cultures. (N-P) Note that activation of Wnt signaling by 6‐BIO treatment aggravated the decrease in LGR5+ cells in irradiated cultures. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.
Figure 4A. Histograms of observed crossovers per F2 individual in the indicated wild type and msh2 populations, from Col×Ct, Col×Ler and Col×CLC crosses (blue). The Poisson expectation is plotted in red. Data from HEI10-OE, recq4a recq4b and HEI10-OE recq4a recq4b F2 populations are also shown for comparison (Serra et al, 2018b; Ziolkowski et al, 2017). Mean values are indicated by black dotted lines. B. Crossovers per 200 kb per F2 plotted along the Arabidopsis chromosomes, with mean values shown by horizontal dashed lines. Data are shown for wild type (red) and msh2 (blue) crossovers generated from Col×Ct, Col×Ler and Col×CLC hybrids. SNPs per 200 kb are shaded in colour (blue=Col/Ct, green=Col/Ler, pink=Col/Cvi). The positions of telomeres (TEL) and centromeres (CEN) are labeled. The number of crossovers analysed is printed inset. C. Data as for B, but analyzing crossovers in wild type (red) and msh2 (blue), or SNPs along proportionally scaled chromosome arms orientated from telomeres (TEL) to centromeres (CEN). Crossover data from HEI10-OE, recq4a recq4b and HEI10-OE recq4a recq4b Col×Ler F2 populations are also shown (Serra et al, 2018b; Ziolkowski et al, 2017).