EMBO/sd-character-level-ner huggingface dataset:
Figure.Figure 3(A) Heatmap showing the differential expression of a selection of ECM-related genes, cytoskeleton and myofibroblast markers in 3 distinct cell lines (M238P, UACC62P, M229P) transfected with the indicated mimics (control (miR-neg), miR-143 or miR-145 mimics, 72 h, 30 nM), assessed by RT-qPCR. (n = 3) (B) M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 μM) in the presence or the absence of LNA-based anti-miR-143 (LNA-143) or anti-miR-145 (LNA-145) (50 nM) or a combination of the two. ECM markers RT-qPCR data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. One-way ANOVA was used for statistical analysis. *P≤0.05, **P≤0.01, ***P≤0.001. (C) Western Blot analysis of ECM remodeling markers on total cell lysates from M238P cells transfected as in (A). (D) Western Blot analysis of ECM remodeling markers on total cell lysates from cells treated with or without BRAFi and the indicated combination of inhibitors (E) Proliferation curves using time-lapse analysis of cells with the IncuCyte system. Graph shows quantification of cell confluence. Two-way ANOVA analysis was used for statistical analysis. ****P≤0.0001. (F) Migration assay performed in Boyden chambers. Representative images showing migrating cells in the different conditions. The histogram represents the quantitative determination of mean ± SD from 6 independent fields representative of 3 independent experiments, using ImageJ software. Paired Student t-test was used for statistical analysis. * P≤0.05, ***P≤0.001. (G) Western Blot analysis of phenotype switch markers on lysates from M238P cells treated as in (A). (H) Crystal violet viability assay of M238P cells treated 72 h with the different combinations of inhibitors in presence or not of BRAFi (Vemurafenib, 3 μM). Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. One-way ANOVA was used for statistical analysis. ****P≤0.0001.
Pol λ also appears to tolerate an primer terminal 8-oxo-dG(syn):dA mispair.Figure 6(A) Substrates used for crystallization of the 8-oxo-dG:dA extension complex. The incoming dNTP (dUMPNPP) is shown in magenta. The primer terminal base (dA) and corresponding templating base (8-oxo-dG) are shown in cyan and orange, respectively. (B) Overview of the 8-oxo-dG:dC extension complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) and an incoming dUMPNPP (magenta). The primer terminal dA and 8-oxo-dG are shown in cyan and magenta, respectively. Key residues that form the primer terminal base pair binding pocket (N513 and R517) and are involved in catalysis (D427, D429 and D490) are shown in black. The nucleotide binding metal (B) is shown in neon. (C) 8-oxo-dG (orange) establishes a Hoogsteen base pair with dA (cyan) at the primer terminus. Tyr505 and Arg517 (purple) interact with the minor groove of the base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ). (D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, incoming dUMPNPP in magenta, and primer terminal dA in cyan. Expectedly, Mg2+ (neon) is occupying the metal B site. However, the metal A is absent in the structure, resulting in a non-catalytic position of the 3′-OH. This is consistent with other structures containing an incoming non-hydrolyzable analog.
Figure 2A: Rates of germinal vesicle breakdown (GVBD) and PB1 emission in oocytes cultured in vitro. Fully-grown GV oocytes were collected from PMSG-primed (44 h) WT and Cnot6l-/- mice. PB1: polar body-1. Error bars, s.e.m. The numbers of analyzed oocytes were indicated (n) B: Representative images of WT, Cnot6l-/- and Btg4-/- oocytes showing PB1 emission at 16 h after culture. Arrows indicate PB1. Scale bar, 100 μm C: Representative images of chromosome spreads made from WT and Cnot6l-/- oocytes after 16 h of in vitro maturation culture. Immunofluorescent staining of topoisomerase II (TOP2) and the centromere antigen CREST were performed to indicate chromosome arms and centromeres, respectively. Numbers of paired sister chromatids are indicated. Scale bar, 5 μm D: Percentage (%) of aneuploidy among in vitro cultured WT and Cnot6l-/- oocytes that have released PB1s. Error bars, s.e.m. *P < 0.05; ***P < 0.001 by two-tailed Student"s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicate E: Rates of GVBD, PB1 emission, and normal spindle assembly in WT, Cnot6l-/- and Btg4-/- oocytes cultured in vitro. Error bars, s.e.m. ***P < 0.001 by two-tailed Student"s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicate F: Confocal microscopy results showing spindle assembly in WT, Cnot6l-/- and Btg4-/- oocytes at metaphase I (MI) and metaphase II (MII). Scale bar, 20 μm G: Pericentrin immunofluorescence showing MTOCs in cultured WT, Cnot6l-/- and Btg4-/- oocytes at MI and MII stages. Spindle and DNA were labeled by microtubule nucleation factor (TPX2) and DAPI, respectively. Scale bar, 20 μm
Figure 3A, B Schematic diagram (A) and experimental setup (B) of BFP assay to characterize force-dependent dissociation kinetics of NKG2D binding with different ligands. C Verification of the binding specificity of NKG2D with different ligands. Data information: Every dot represents the adhesion frequency of one cell-bead pair and the experimental data came from at least two to three repeated trials. Horizontal lines represent mean values. Error bars represent mean ± SEM. D Representative force vs. time curve for measuring single NKG2D-ligand bond lifetime. E, F Force-dependent bond lifetimes of NKG2D and various ligands at 5 pN, 10 pN and 15 pN (E) and full force spectrum (F). Data information: Bond lifetimes in (E) and (F) (in which n = 1505 for MICA, n = 1852 for MICB, n = 531 for ULBP1, n = 1241 for ULBP3) of all ligands with NKG2D came from at least 19 NK cell-bead pairs of at least four independent biological experiments. bars in (E), and data points in (F) represent mean values. Error bars represent mean ± SEM. G Ratios of average bond lifetimes for NKG2D/MICA to that of NKG2D and other ligands.
Hoxa4 KO mice alleviate smooth muscle phenotypic modulation and abrogate neointima formation after carotid artery ligation injury.Figure 7Quantitative real-time PCR analysis of differentiated smooth muscle marker genes in the left carotid artery of 8-week-old WT and Hoxa4 KO mice (WT: n=7, Hoxa4 KO: n=5). Quantitative real-time PCR analysis of differentiated smooth muscle marker genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation (n=6). Mean expression level of six WT mice at the baseline was defined as 1.0. Hoxa4 expression levels in the ligated left carotid artery of WT mice at 1 week after ligation (n=6). Quantitative real-time PCR analysis of Yap/Tead target genes in the left carotid artery of 8-week-old WT and Hoxa4 KO mice (WT: n=7, Hoxa4 KO: n=5). Quantitative real-time PCR analysis of Yap/Tead target genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation (n=6). Mean expression level of six WT mice at the baseline was defined as 1.0. Representative western blotting analysis and densitometry of non-ligated (right; R) and ligated (left; L) carotid arteries from WT and Hoxa4 KO mice at 1 week after ligation (WT: n=6, Hoxa4 KO: n=7). *P<0.05, **P<0.01, by two-way ANOVA with Sidak's correction. Representative images showing Elastica van Gieson staining (G), and vessel, neointima and media area (H), and neointima to media layer ratio (I) of carotid arteries of WT and Hoxa4 KO mice at 4 weeks after ligation (WT: n=14, Hoxa4 KO: n=11).
In vivo and in vitro proteomics-based assays link NlpI to several classes of PG hydrolases.Figure 1A-B. Wild-type and ΔnlpI cells were heated at a range of temperatures and the soluble components were labelled by TMT, combined and quantified by LC-MS, using the published 2D-TPP protocol (Mateus et al., 2018). Shown are volcano plots of two replicates depicting changes in: protein abundance (A) and thermostability (B). A local FDR (false discovery rate) <0.01 was set as a threshold for significance. Highlighted proteins: outer membrane proteins (OMPs, light green), β-barrel assembly machinery (BAMs, red), PG synthases/regulators (green), PG hydrolases and regulators (blue) and the Tol-Pal complex (violet). All other proteins were colored grey and not labelled to increase the plot clarity. C-D. Affinity chromatography with immobilised NlpI. Membrane extracts from E. coli were incubated in low and high salt binding conditions (50 and 400 mM NaCl, respectively), and then eluted with 1 M NaCl or 2 M NaCl to identify possible interaction partners by label-free LC-MS analysis. The plot shows the log2 fold enriched proteins when compared to those eluted from a parallel empty column control, versus the log10 p-value, in low (4 replicates) (C) and high (2 replicates) (D) salt. Highlighted points are all interactions with PG enzymes and their regulators, as well as members of the divisome. All other proteins were colored grey and not labelled to increase the plot clarity; many were non-physiological interactions with abundant cytoplasmic proteins.
Targeted delivery of R-III to hepatic stellate cells.Figure 7Liver sections from treated mice were immunohistochemically stained for desmin, His-tag, and α-SMA. His-positive signals largely overlapped with desmin/α-SMA staining. Scale bar, 50 μm.
Figure 4A, B. Distribution of the max CADD38 PHRED (model for GRCh38) scores for (A) cysteine (n=1,401) and (B) lysine (n=4,363) CpDAAs of low, medium, and high intrinsic reactivities, defined by isoTOP-ABPP ratios, low (R10:1>5), medium (2<R10:1<5), high (R<2) Kruskal-Wallis nonparametric test to examine reactivity group difference, Wilcox test used for pairwise comparisons (BH-adjusted p-values, *p. adj = 0.04, **p. adj = 0.0037, ***p. adj = 0.00013). The boxplot boxes represent the lower and upper quartiles, with the central band as median. Notches show the confidence interval based on median ± 1.58*IQR/sqrt(n), and whiskers mark observations that satisfy quartiles ± 1.5*IQR. Median CADD38 max codon scores with bootstrapped 95% confidence intervals for reactive groups are: low CpD Cys 27.3 [26.9, 28.0], medium CpD Cys 28.55 [27.80, 29.05], high CpD Cys 31 [28.8, 32.0], low CpD Lys 29.5 [29.3, 29.6], medium CpD Lys 29.25 [28.85, 29.50], high CpD Lys 29.05 [28.50, 29.55]. C. Shows CADD38 max codon scores for nonsynonymous SNVs at residues 220-479 of CASP8 (UniProt ID Q14790). Dashed horizontal line marks deleterious threshold of 25. D. Crystal structure of CASP8 (PDB ID: 3KJN) highlighting C360 and C409. Bound covalent inhibitor B93 in yellow, with distance between detected cysteines and inhibitor measured in Angstroms. Protein surface color represents CADD38 max codon scores. Image generated in PyMOL E. Activity of recombinant caspase-8 protein assayed using fluorogenic IETD-AFC substrate. Percentage activity shown relative to wild-type (WT) protein for three replicate experiments, bars and error bars as mean ± SD.
Quantitative analysis of nucleotides, nucleosides, and bases at their destinations A Schematic representation of reactions of the nucleotide degradation pathway.Figure 6A Schematic representation of reactions of the nucleotide degradation pathway. B, C Nucleosides (B) and nucleobases (C) within the cells. Wild‐type and atg2∆ were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, the cells were collected; their metabolites were extracted, and the nucleosides and nucleobases were analyzed by LC/MS. The level of each nucleoside and base is presented as its absolute concentration, as described in and Supplementary Methods. All data are means of duplicates except the wild‐type strain at 1 h. D, E Nucleosides (D) and nucleobases (E) in the medium. The culture medium of the above cultures was filtered, and nucleosides and nucleobases were directly analyzed by LC/MS as described in and Supplementary Methods. All data are means of duplicates. F, G Destinations of nucleosides and bases. Based on the data from (B-E), the level of each nucleoside and base in cells (open circle) and in cells and medium (closed circle) of wild‐type strain were estimated. See Supplementary Methods in detail (flow chart of the calculation procedure).
Figure 4(A) WT and SKO cells stably expressing ACSL3-Cherry were transfected with ADRP-GFP for 24 h, incubated with OA for 1 h, fixed and stained with LD540. Single confocal planes of deconvolved confocal z-stacks. (B) Colocalization analysis of (A), from single confocal sections. Bars: correlation coefficient m1 ± SEM, n=34-35 cells/genotype, 2 experiments, **p<0.005 (unpaired T-test). (C) WT and SKO cells were transfected with ADRP-GFP for 24 h, incubated with OA for 2 h and co-plated with delipidated WT or SKO cells stably expressing ACSL3-Cherry. After co-plating, cells were incubated for 2.5 h in normal growth medium, then fused together using PEG. Cells were fixed and imaged 1 h post-fusion. Single confocal planes. (D) Colocalization analysis of (C). Bars: correlation coefficient m1 ± SEM, n=31-39 cells/treatment, 3 experiments, **p<0.005 (Tukey's test). Similar results were obtained when cells were kept in delipidation medium after co-plating. (E) WT and SKO cells were treated with OA for 1 h, fixed and stained with anti-ACSL3 antibodies and LD540. Representative sections of peripheral ER areas, single confocal planes of deconvolved z-stacks. (F) Analysis of (E), insets illustrate assigned categories of ACSL3LD association. Bars: % of LDs/ROI in the indicated category, mean ± SEM, n= 9-10 peripheral ROIs from 9-10 cells/treatment (616-968 LDs), 3 experiments, **p<0.005 (unpaired T-test). (G) TEM images of anti-ACSL3-labeled WT and SKO cells treated with OA for 1 h. (H) Analysis of (G), n= 66-119 LDs from 8 cells/genotype.
Figure 2(A) Bright-field images (10x (left) and 4x (right) magnification) from shScr, sh1 and sh2 at day 8 of differentiation. Scale bars, 200 µM. (B) quantification of embryoid bodies (EB) generated from shScr, sh1 and sh2 at day 8 of differentiation. Scale bars, 200 µM. C. Quantification of BrdU incorporation in shScr, sh1 and sh2 ESCs at the indicated days of EB differentiation. Data are relative to shScr. D. RT-qPCR of Zfp207, the pluripotency genes (Oct4, Nanog and Sox2), the mesodermal markers (Msx1 and Brachyury, T), the endodermal markers (Foxa2, Sox17), and the neural-associated genes (Pax6, Sox11 and Nestin) in shScr, sh1 and sh2 ESCs along the time-course of EB-mediated differentiation. mRNA levels are relative to the expression of shScr at day 0.
Effector and immunity combinations alter the VpT6SS1-based platform's toxicity range.Figure 5A-D (A, B, D) Viability counts of V. parahaemolyticus RIMD 2210633 ∆hcp1 (A), V. alginolyticus 12G01 ∆hcp1/∆hcp2 (B), and V. natriegens (D) prey bacteria before (0 h) and after (4 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose at 28 °C. Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. ( C) Illustration summarizing the competitions shown in (A, B). Effector and immunity modules expressed by VnatReg carrying pT6SSEffectorless (yellow) are colored to match the bacterium from which they were derived.
Figure 4A-C, PIL1, AT5G02580 and CDF5 expression pattern in 3-d-old SD-grown seedlings from indicated plant lines at night. Data is representative of 3 biological trials. D-F, Expression of PIL1, AT5G02580 and CDF5 in 3-d-old SD-grown seedlings at ZT18. 5 biological trials were averaged, error bars indicate SEM. Different letters denote statistically significant differences based on Wilcoxon test (p<0.05).
NMDAR activation requires conformational mobility at inter-domain and inter-layer interfaces.Figure 1Schematic representation of a full-length GluN1/GluN2B NMDAR. The receptor displays a layered architecture. NTD, N-terminal domain; ABD; agonist-binding domain; TMD, transmembrane domain. The studied inter-domain and inter-layer interfaces are reported with numbered yellow stars. For clarity, interface 4 is shown from a side view (inset) Representative current traces from oocytes expressing wild-type (WT) and double cysteine mutant NMDARs before and after DTE treatment. For each mutant, traces are normalized (in height, not in current amplitude) to the maximal response obtained after DTE treatment on wild-type (WT) receptors Summary of the DTE-induced potentiation of WT and double cysteine mutants Immunoblots from Xenopus oocytes expressing either wt or mutant subunits. M1 indicates the GluN1 monomer (~110 kDa); M2 the GluN2B monomer (~180 kDa); D1/1 the GluN1 homodimer (~220 kDa); and D1/2 the GluN1/GluN2B heterodimer (~290 kDa). Lower panels: immunoblots in reducing conditions (+ β-mercaptoethanol). N.I., non-injected oocytes
Figure 7(a) Top: Schematic representation of the IgH locus depicting Sμ and Sα region with the position of guide RNAs (gRNA) that were used to induce blunt end DSBs using WT SpCas9. Bottom: IgH locus depicting Sμ and Sα region with the position of guide RNAs (gRNA) that were used to induce staggered DSBs with 5′ overhangs using D10N mutants of SpCas9 (Cas9n). (b) FACS profile of the percentage of WT and Samhd1 KO CH12F3-2A cells undergoing IgA switching following transfection with the indicated plasmids. The number in the FACS plot represents the percentage of cells expressing IgA on their surface. (c) Summary of the FACS data obtained from two independent experiments. The data are shown as percentage of IgA expressing cells upon CSR induced by blunt (left) or staggered (right) DSBs (n = 2; mean + sd; two-tailed unpaired Student's t-test); ns = not significant. (d) Length of resected DNA at Sμ-Sα junctions obtained from the joining of DSBs with blunt ends. Statistical significance as evaluated by two-tailed unpaired Student's t-test is shown. (e) Summary of the characteristics of Sμ-Sα recombination junctions resulting from the joining of DSBs with blunt ends. (f) Length of resected DNA at Sμ-Sα junctions obtained from the joining of staggered DSBs. Left: Length of Sμ and Sα overhangs were 35 nt and 98 nt, respectively. Right: Length of Sμ and Sα overhangs were 35 nt and 187 nt, respectively. (g) Summary of the characteristics of Sμ-Sα recombination junctions resulting from the joining of staggered DSBs. Statistical significance as evaluated by Fisher's exact test is shown. (h) Summary of the average length of insertions at Sμ-Sα recombination junctions. Statistical significance as evaluated by two-tailed unpaired Student's t-test is shown. (i) Sequences of Sμ-Sα recombination junctions with insertions. The middle column indicates the insertions (in) found in different clones at the junctions. Analysis of the effect of TdT expression on the frequency of CSR as well as characteristics of Sμ-Sα recombination junctions resulting from the joining of DSBs with blunt ends. (j) Top: Schematic representation of the experimental design to analyze the effect of TdT expression on CSR in WT and Samhd1 KO cells. Bottom: Western blot analysis of the expression of TdT in WT and Samhd1 KO cells. Tubulin was used as a loading control. (k) FACS profile of the percentage IgA switching in WT or Samhd1 KO CH12F3-2A cells with or without TdT expression. (l) Summary of the FACS data obtained from two independent experiments (n = 2; mean + sd; two-tailed unpaired Student's t-test; * = p ≤ 0.05). (m) Summary of the characteristics of Sμ-Sα recombination junctions. Statistical significance as evaluated by Fisher's exact test is shown. ns = not significant.
Figure 3(A) Venn diagrams of differentially expressed genes following comparison of AhR KO (KO/WT) and TCDD effects (TCDD/vehicle in WT mice) in sorted stem and progenitor cells; KO (HGC mice); WT (GC mice). An FDR<0.1 cutoff was used. (B) Heatmap comparison of the top 25 enriched Diseases and Biological Functions in AhR KO (KO/WT) and TCDD (TCDD/vehicle in WT mice) treated cells as identified by IPA. (C) Heatmap comparison of the top 25 upstream regulators between AhR KO (KO/WT) and TCDD (TCDD/vehicle in WT mice) treated cells. (D-G) Gene Set Enrichment Analysis (GSEA) was performed in sorted stem and progenitor cells from AhR WT (n=3) and KO (n=4) mice (D&E) and in WT mice in response to vehicle and TCDD treatment (F&G). Normalized enrichment scores (NES) and FDRs were calculated using the Public Interaction Database (PID) for the FoxM1 Pathway gene set.
Figure 2(A) SKO cells stably expressing WT-seipin-GFP cells were delipidated and then incubated with OA and LipidTox Deep Red for 30-75 min. Airyscan video snapshots, upper panel: an example of 2 seipin-GPF punctae, one stably associated with a LD (blue arrowhead) and another not LD associated (orange arrowhead). Lower panel: an example of a mobile LD, with a single seipin puncta moving together with it (blue arrowhead). (B) Analysis of (A), bars: mean velocity of tracked seipin puncta ± SEM, n=13 ROIs from 13 cells, 90 puncta, 2 experiments, **p<0.005 (unpaired T-test). (C) Analysis of LD mobility in WT and SKO cells. Cells were incubated with OA for 45-75 min, LDs were visualized with LD540 and cells imaged by widefield microscopy. Bars: 1 - [Pearson's colocalization coefficient between subsequent frames/cell], ± SEM, n= 149-151 cells, 2 experiments, **p<0.005 (Mann Whitney test). (D) Examples of LD trajectories from cells treated and imaged , arrowhead indicates the tracked LD. (E) Analysis of the LD mobility from (C) and (D), individual LDs plotted for volume and mean velocity. (F) WT and SKO cells were transfected with ER marker plasmid (sfGFP-ER-3, green), delipidated overnight and incubated with OA and LipidTox Deep Red (magenta) for 15-75 min. Airyscan video snapshots exemplifying different modes of LDER-associated movement, note the different time scales. Panel I and II: WT cell, panel III: SKO cell. (G) Analysis of LDER-associated movement from live airyscan microscopy as in (F). Bars: % of LDs/ROI displaying indicated mode of movement, ± SEM, n= 43-55 ROIs (201-467 LDs) from 4 experiments, **p<0.005 (Mann Whitney test). (H) Analysis of LD velocity. Bars: mean velocity of tracked LDs, ± SEM, n=29-42 LDs/group, 2 experiments, **p<0.005 (Kruskal-Wallis test, followed by Dunn test). (I) WT and SKO cells stably expressing HSP47-APEX were delipidated, treated with OA for 1 h and processed for SB-EM. 3D models of LDs and surrounding ER and mitochondria profiles shown on top of a block face image from SB-EM dataset. Insets are representative cross sections from modeled area highlighting the ER-LD association. Red= LD, Yellow= ER, Green=mitochondria. (J) Analysis of LDs from (I). Black lines indicate median, boxes third quartiles, n= 98-227 LDs. (K) Analysis of LDs displaying no ER contacts from (I). Bars: mean % of LDs in ROIs not in proximity to the ER, ± SEM, n= 11-16 ROIs/genotype, **p<0.005 (unpaired T-test).