EMBO/sd-character-level-ner huggingface dataset:
Figure 5A Alkaline gel Southern blot analysis of AAV DNA. AAV DNA was hybridized to a probe specific for the HLP promoter. Neg, AAV DNA treated with Dnase I; CodopV3, AAV-CodopV3; I, AAV-5'N6-N-intein; II, AAV-C-intein-3' N6. B Chromogenic assay performed on plasma samples to detect F8 activity in AAV-treated mice compared to controls groups. Data are presented as mean ±SEM. The same animals were analysed at the various time-points. Each dot within the same group and within different groups of treatment corresponds to a single animal. The baseline includes all animals (n=20) before the treatment: CodopV3: n=8; N6 intein: n=5; CodopN6 intein: n=5. The statistical difference between groups has been assessed at 4weeks post-injection (wpi) with the One-way Anova p value = 0.056; at 8 wpi with the Kruskal-Wallis test p value = 0.68; at 12wpi with the Kruskal-Wallis test p value = 0.45; at 16wpi with the Kruskal-Wallis test p value = 0.58. C Activated partial thromboplastin time (aPTT) assay performed on plasma samples both at the baseline and at the last time-point of the analysis (16 wpi). Data are presented as mean ±SEM. Each dot within each group of treatment corresponds to different animals: The baseline includes all animals before the treatment n=21; CodopV3: n=8; N6 intein: n=5; CodopN6 intein: n=5. Significant differences between groups were assessed using the Kruskal-Wallis test. **indicates the significant difference between the baseline and the N6 intein groups: p value ≤ 0.01.*indicates the significant difference between the baseline and the CodopN6 intein groups: p value ≤ 0.05. D Tail-clip assay reported as bleeding time in seconds. Significant differences between groups were assessed using One-way Anova test p value is = 0.11.Data are presented as mean±SEM. Each dot corresponds to a different animal. Knockout (K.O.) group: n=4; CodopV3: n=4; N6 intein: n=4; CodopN6 intein: n= E Haemoglobin content measured as Optical Density (OD) at 416 nm after the tail-clip assay. Significant differences between groups were assessed using One-way Anova test. Data are reported as mean±SEM. Each dot corresponds to a single animal. Knockout (K.O.) group: n=4; CodopV3: n=4; N6 intein: n=4; CodopN6 intein: n=6. F Western blot analysis of liver lysates (100 µg) from either CodopN6 intein-treated mice (n=6) or untreated haemophilic mice (n=2). A lysate from HEK293 cells transfected with the N6 full-length plasmid was used as positive control (50 µg). Arrows point at N6 full-length protein (N6), the 5' half of N6 (5'N6) and 3' half of N6 (3'N6).
Fig.Figure 5Flow cytometry analysis of Mitotracker (MT) Green and Red integration in Gata6-WT and Gata6-KOmye naïve pMɸ (gated on F4/80hi/+ Tim4+) in vivo, 30 min after intraperitoneal injection (i.p.) of 1 µM of Mitotracker Green and Red. Data shown are representative of 3 to 5 independent mice of each genotype and are expressed as mean +/- SEM. Student's t test analysis was performed. Flow cytometry analysis of Mitotracker green and red staining in Gata6-WT and Gata6-KOmye pMɸ (gated on F4/80hi/+ Tim4+) unstimulated (-) or after LPS (100 ng.ml-1), IL-10 (10 ng.ml-1), αIL-10R (5 µg.ml-1) or isotype (5 µg.ml-1) stimulation for 16 h in vitro. Data shown are pooled from 3 independent experiments and normalized to WT unstimulated. Flow cytometry analysis of MitoSOX staining in Gata6-WT and Gata6-KOmye pMɸ (gated on F4/80hi/+ Tim4+) unstimulated (-) or after LPS (100 ng.ml-1), IL-10 (10 ng.ml-1) and αIL-10R (5 µg.ml-1) stimulation for 16 h in vitro. Data shown are pooled from 2 independent experiments and normalized to WT unstimulated. IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 500 µM MitoTempo, 10 mM NAC, 0.5 µM MitoQ for 16 h. Data are representative of at least 3 experiments.
Smaducin-6 shows bactericidal effects through neutrophil recruitment.Figure 6Subcutaneous injection of Smaducin-6 into CLP mice reduced bacterial loads in peritoneal fluid. n = 10 mice per group per experiment. Data were statistically analyzed by the Mann-Whitney U-test. **P < 0.005 compared to sham or vehicle control (CLP + Pal-Scram #1). Smaducin-6 increases neutrophil numbers in peritoneal lavage fluids from LPS-induced endotoxemia BALB/c mice. n = 5 mice per group per experiment. Data were statistically analyzed by a t-test. **P < 0.005 compared to sham or vehicle control (CLP + Pal-Scram #1). CXCR2 expression in human neutrophils, measured by FACS, was decreased by LPS treatment and restored by Smaducin-6 treatment. Treatment with Smaducin-6 downregulates LPS-induced GRK2 expression in RAW264.7 cells and human neutrophils. CXCR2-expressing neutrophils isolated from CLP mice in the presence of Smaducin-6 or Pal-Scram peptide were analyzed by FACS. n = 5 mice per group were used. Data were statistically analyzed by a t-test and show the mean ± SD of at least three independent experiments. **P < 0.005 compared to sham or vehicle controls (CLP + PBS and CLP + Pal-Scram #1). GRK2 expression was directly analyzed by immunoblotting in neutrophils isolated from CLP mice in the presence of Smaducin-6 or Pal-Scram #1 peptide.
Figure 6(A) Diagram to show the extent of flank DNA binding for RAG and HzTransib in the STC. Red, target site DNA; light pink, flank DNA bound by both RAG1 and HzTransib; blue, remainder of flank DNA, much of which is contacted by RAG2 in the RAG STC. (B) Left, structure of Paired RAG STC with RAG2 omitted (top view). RAG1 protein is shown as an electrostatic surface and target DNA in cartoon mode with the backbone colored yellow and base colored cyan. The white arrow indicates the positively charged surface for target DNA binding on RAG1. Right, structure of the Paired RAG STC with one RAG2 shown (45° rotation relative to (A)). RAG1 is shown as a molecular surface and colored white. RAG2 is shown as an electrostatic surface. The white arrow indicates the positively charged surface for target DNA binding on RAG2. (C) HzTransib STC structure is shown as for RAG in (B). Three white arrows indicate the positively charged surface provided by HzTransib for target DNA binding. (D) Front view of HzTransib STC near the integration site (PDB:6PR5), depicted as in Fig 3D. (E,F) Protein electrostatic surface around M847/M848 of RAG1 in the Paired STC and equivalent region of HzTransib STC. Loop region rendered transparent. Dashed line, cavity formed due to shorter loop in RAG1. (G) A similar view of RAG Dynamic STC as shown in (E) depicting bases C-t-2 and G-t-1 which are flipped and project into the cavity below target site DNA in the RAG STC.
Figure 5RAD21 and EMT-related molecules in parent MDA-MB-453breast cancer cells (P) and the corresponding CSLCs (indicated by CSC or CSLC) sorted according to the CD44+ cancer stem cell markers were observed by (A) qPCR. Data are presented as the mean ± S.D. for three independent experiments. *P < 0.01. Statistical significance was validated by Student's t-tests. RAD21 and EMT-related molecules in parent MDA-MB-453 breast cancer cells (P) and the corresponding CSLCs (indicated by CSC or CSLC) sorted according to the CD44+ cancer stem cell markers were observed by (B) Western blot analysis. Data are presented as the mean ± S.D. for three independent experiments. *P < 0.01. Statistical significance was validated by Student's t-tests. (C) Cell migration assay of MDA-MB-453 parental and CSCL cells using Transwell chamber without matrigel. Graphs indicate the average number of cells per field. *P < 0.01. Statistical significance was validated by Student's t-tests. (D) Relative crosslinking frequencies among CTCF/RAD21 binding sites on the genes were measured with a 3C assay in the parental cells (blue line) or CSLCs (red line). BamH1 or Xba I restriction sites on the TGFB1or ITGA5 gene, respectively, appear as gray shaded bars. Black shading indicates the anchor fragment. Each value was normalized to the crosslinking frequency at the ERCC3 gene. The maximum crosslinking frequency was set at 1 (mean ± S.E.M, n = 3). *P < 0.01, **P <0.05. Statistical significance was validated by Student's t-tests. (E) A ChIP assay was performed for the CSLCs (red bar) or parental cells (blue bar) using antibodies specific for RAD21, AcH3, Pol II, or IgG (as the control). Enrichment was measured by qPCR and reported relative to the total input (4%). ChIP signals for AcH3 was normalized to total H3. Results for at least three chromatin preparations are shown ± S.E.M. *P < 0.01, **P <0.05. Statistical significance was validated by Student's t-tests.
Figure 2A) Inspection of specific amphid-neuron-defective mutants revealed that AWA neurons are most likely responsible for pheromone perception. Here, an edge connects neurons to a genetic mutant if the neurons are documented to be affected in the mutant. The color code of edges represents the average C.I. of multiple genetic mutants with impaired neuronal functions or specified abnormal neuron identities. The region colored green shows the range of wild-type sex pheromone response in the male. 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) AWA-laser-ablated males were not attracted by the sex pheromone in single-worm chemoattraction assays. Both AWA-defective mutant males (lin-11 and odr-7) and AWA-laser-ablated males failed to reach the sex pheromone within 30 min. Wild-type C. elegans males responded normally to the sex pheromone and wild-type C. elegans hermaphrodites were not attracted by same-sex pheromone. The single-worm chemoattraction assay was performed as described in Materials and Methods. Sample size of males in AWA-laser-ablation experiment: 12; all other experiments: 100 worms.
GlaB inhibits Gli1‐dependent growth of cerebellum‐derived normal progenitors A qRT–PCR shows Hh target mRNA expression levels determined in 6‐day‐old mouse cerebellar progenitors after s.Figure 4A qRT-PCR shows Hh target mRNA expression levels determined in 6‐day‐old mouse cerebellar progenitors after s.c. injections of GlaB. B Representative EGLHE staining of 6‐day‐old mousecerebellum after s.c. injections of GlaB (upper panel); immunohistochemistry shows Gli1 (middle panel) and Ki67 stainings (lower panel). In all images, the scale bar represents 50 μm. C The graph shows the percentage of EGL thickness reduction in GlaB‐treated mouse cerebella in comparison with vehicle mouse cerebella. Data show the mean ± SD of cerebella (n = 10) for each treatment. *P 0.05 versus Ctr. D BrdU assay in GCPs. Cerebellargranule cell progenitors (GCPs) isolated from 4‐day‐old mice were treated with Shh (recombinant mouseSonic Hedgehog, amino‐terminal peptide, ShhN, 3 μg/ml) and with different concentrations of GlaB, as indicated, for 48 h. Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO‐treated sample. Shown is the mean of three independent experiments. Error bars indicate SD. *P 0.05 Shh versus DMSO; **P 0.05 Shh + GlaB versus Shh. E qRT-PCR and show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6‐day‐old mousecerebella after s.c. injections of GlaB. In all qRT-PCR experiments, the results were normalized to endogenous control (β2‐microglobulin and HPRT). *P 0.05 versus Ctr. F Western blot analysis show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6‐day‐old mousecerebella after s.c. injections of GlaB.
Figure 4(A-C) Graphic representation of the surface densities and the nanoscale distributions of CD22, CD19, IgM and IgD.(A) Size estimates for IgD/M and CD19/CD22 (left) and random distribution (centre and right) of the number of molecules measured in Figure 2A.(B) Surface distribution of the actual numbers of CD22, CD19, IgM and IgD, taking into consideration their clustering tendency. Full 1x1 µm2 view (top) and zoom into black dashed square (bottom).(C) Integration of the distributions of CD22, CD19, IgM and IgD (from Figure 4B) into one graph. Full 1x1 µm2 view (top) and zoom into black dashed square (bottom). (D-F) In silico model and simulation of area covered by CD22 nanocluster diffusion.(D) SIM and dSTORM precision images of IgD (top) and CD22 (bottom) to show every localization of IgD (in red) and CD22 (in blue). Images were then overlaid to indicate areas (green) where CD22 (blue) overlaps with IgD (red), highlighting the areas where IgD would be overlapping with CD22 (top right) and where CD22 would be overlapping with IgD (bottom right).(E) Computer simulation of area covered by diffusing CD22 nanoclusters. CD22 nanoclusters were distributed in a simulated 1x1 µm area according to clustering parameters from dSTORM. Nanoclusters were then allowed to move with the indicated lateral mobilities over time. The total area covered (in per cent) by CD22 is indicated when reaching 500 ms.(F) Area covered by CD22 nanoclusters diffusing with 0.005 µm2/s (slow) or 0.100 µm2/s (fast).
Inhibition of the fission machinery or of Bnip3 partially protects from muscle loss.Figure 3(A) RNAi‐mediated knockdown of Fis1 showed by western blot assay. MEF cells were transfected with vectors expressing four different Fis shRNAs. For in vivo experiments Fis (4) was used. Efficiency of protein knockdown is indicated in Supplementary Table S2. (B) Blockade of Fis1 and of Bnip3 by RNAi reduces muscle loss during fasting. Adult skeletal muscles were transfected with specific shRNAs for mouse Fis1 (oligo 4) and mouse Bnip3 or co‐transfected and 8 days later mice were starved for 48 h. Cross‐sectional area (CSA) of transfected fibres, identified by GFP, was measured as described earlier (*P0.01) n=450. (C) RNAi of Fis1 and Bnip3 significantly prevents MuRF‐1 and atrogin‐1 promoters activation during starvation. TA muscles were transfected with either 5 kb of the MuRF‐1 promoter (left panel) or 3.5 kb of atrogin‐1 promoter (right panel) with or without shRNAs vectors against Fis and Bnip3. In fasting experiments, after 7 days from transfection mice were starved for 24 h. A renilla‐luciferase construct (pRL‐TK) was co‐transfected to normalized for transfection efficiency. The activation of MuRF‐1 and atrogin‐1 promoters was determined by firefly activity and normalize for the renilla. Each condition represents the average of at least three independent experiments±s.e.m. (*P0.05). (D) Muscle atrophy induced by FoxO3 is reduced by d.n.DRP1. Adult skeletal muscles were transfected with HA‐tagged c.a.FoxO3 with or without d.n.DRP1 and examined after 2 weeks. CSA of transfected fibres, identified by anti‐HA immunofluorescence, was measured as described earlier (*P0.001) n=400. (E) Blockade of the fission machinery by d.n.DRP1 or Bnip3 inhibition by RNAi reduces FoxO3‐mediated autophagosome formation. Animals were transfected with YFP‐LC3, c.a.FoxO3 in presence or absence of d.n.DRP1 or shRNAs against Bnip3. Twelve days later, muscles were collected and analysed for fluorescent vesicles formation (*P0.001) n=450. (F) FoxO3‐dependent activation of MuRF‐1 and atrogin‐1 is significantly reduced by inhibition of the fission machinery. MuRF‐1 promoter (left panel) or atrogin‐1 promoter (right panel) was co‐transfected into adult TA muscle with or without c.a.FoxO3 and in the presence or absence of d.n.DRP1. A renilla‐luciferase vector was co‐transfected to normalize for transfection efficiency. Eight days later, firefly and renilla‐luciferase activity was determined. Each condition represents the average of at least three independent experiments±s.e.m. (*P0.05).
Figure 1(A) The Venn diagram depicts the overlapping of histone mRNAs detected in a previously performed rRNA-depleted nuclear RNA-seq (RPM>1) and in ALYREF-iCLIP-seq (with clustered binding sites in at least two out of three biological replicates). (B) The ratio of unique histone iCLIP-seq read population to unique histone RNA-seq read population was calculated and shown, with the unique histone RNA-seq read population set as "1". Error bars represent standard deviations from three replicates of ALYREF iCLIP data. Statistical analysis was performed using Student's t-test. ***P< 0.001. (C) ALYREF RIP-qPCRs to examine ALYREF binding on histone mRNAs. Relative RIP efficiencies are shown. A tRNA was used as a negative control for ALYREF binding. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. **P < 0.01, ***P< 0.001, n.s., not significant. (D) The distribution of histone reads in 5' UTR, CDS and 3' UTR in rRNA-depleted nuclear RNA-seq and ALYREF-iCLIP-seq. (E) Metagene analysis of normalized ALYREF signals on histone mRNA based on iCLIP-seq data. iCLIP-seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA's contribution to the plot. (F) Screenshots of two histone mRNAs showing clustered ALYREF binding sites. The 3' UTR regions are highlighted. (G) Distribution profile of clustered ALYREF binding sites on histone mRNA 3' UTRs relative to the cleavage site (CS). The CS is set as "0" in the x axis, and the relative position to the CS is marked. The y axis shows the total occurrence of binding sites in three repeats at each nucleotide. (H) Graphic displays ALYREF binds next to SLBP on histone mRNAs.
Endogenous presentation of NeoR on MHC class II does not depend on components of the MHC class I presentation pathway.Figure 2(A), pINCO‐NeoR transfected EBV‐B1.11 cells (B), and as control pINCO‐Tyrosinase‐transfected EBV‐B1.11 cells (C), were either left untreated or treated with 5 μM lactacystin or 50 μM AAF‐CMK for 20 h. Following fixation with 0.5% paraformaldehyde, cells were incubated with T cells, and 24 h later GM‐CSF release was determined by ELISA. (D) FACS analysis of EBV‐B1.11 cells (open histograms) and EBV‐B1.11 cells stably transfected with the ICP47 gene (shaded histograms). Inhibition of TAP by ICP47 down‐regulates HLA class I but not HLA‐DP expression. An isotype‐matched antibody was used as control (thin‐lined histogram). EBV‐B1.11‐ICP47 cells were transfected with pINCO‐NeoR (E), pINCO‐Tyrosinase (F), or mock transfected (pINCO) and MHC‐restricted presentation monitored with T cells.
Fig.Figure 6Deletion of VE-cadherin was induced in adult mice (13 weeks) and EdU incorporation into PROX1-positive cell nuclei was determined 6 weeks after Tamoxifen administration. Shown are mesenteric wholemount stainings prepared from control (A, B; n=3) or VE-cadherin-deleted mice (C, D; n=3). (B) and (D) show a magnification of the area outlined by the white dashed line in (A) and (C). Antigens shown in addition to the EdU incorporation are depicted in colour on the left side of the first panel. PEC, PECAM1; PRX1, PROX1. Scale bars = 100 µm Enumeration of EdU-positive LECs. The measurements were obtained from three animals and nine confocal stacks per biological group. PROX1-positive nuclei were counted using the particle analysis tool of Fiji. Co-localization of PROX1 and EdU was determined manually in each confocal plane, using the cell counting tool of Fiji. The data represent mean ± SD. **** p ≤ 0.0001. Two-tailed unpaired Student's t test Aberrantly enhanced VEGFR-3 phosphorylation in VE-cadherin-deficient mesenteric LECs of six weeks old mice that had received 4-OHT injections at P2 and P4. Shown are wholemount immunostainings for the antigens shown on the left in color. pVR3, pVEGFR-3, PRX1, PROX1; PEC1, PECAM1. (G) and (I) show a magnification of the area outlined by the white dashed box in (F) and (H). Scale bars = 50 μm. The data are representative for 6 (Cdh5lox/lox) and 5 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals from 2 litters Dermal, mesenteric and intestinal tissue pieces of 15 week old control (Cdh5lox/lox) mice were dissociated and total RNA was extracted immediately. For reverse transcription 2.0 µg of total RNA was used for each tissue type. Expression of lymphangiogenic factors VEGF-C and VEGF-D was determined by quantitative real-time (qRT)-PCR using specific TaqMan probes. Samples were measured in triplicate and diagrams are representative for 3 independent experiments. Relative expression (ΔΔCt) of target genes was normalized to the control transcript UBC Loss of VEGFR-3 surface expression on mesenteric LECs growing in cell sheets was associated with concomitant upregulation of VEGFR-2, scale bars = 50 μm. The data are representative for 4 analyzed animals from 2 litters.VR2, VEGFR-2; VR3, VEGFR-3, PRX1, PROX1 Expression of the transcriptional co-activators YAP and TAZ was analyzed in mesenteric wholemount preparations 8 weeks after induction of VE-Cadherin deletion at 1 year of age by three doses of Tamoxifen. Respective antigens depicted are indicated on the top of the panels in colour. The highly related molecules YAP and TAZ were both recognized by the antibody used and are therefore collectively abbreviated Y/T. Scale bars = 100 μm PROX1+ nuclei (red in panels M and N) were classified into YAP/TAZ low, intermediate and high-expressing cells, according to their relative YAP/TAZ immunoreactivity in wildtype and VE-cadherin-deleted mice. The data were obtained from three animals per biological group. PROX1-positive and YAP/TAZ-positive nuclei were counted manually. The pie charts represent mean values
Figure 3Left: Data generated as described above in Figure 2 was utilized for Gene Set Enrichment Analysis (GSEA) Molecular Signature DataBases (MSigDB) KEGG analyses. Cut-off for reporting was a false discovery rate q value of < 0.25, and normalized enrichment scores (NES) are shown, with darker colors indicating more enrichment. Middle: Data generated as described above in Figure 3 was utilized for Gene Set Enrichment Analysis (GSEA) Molecular Signature DataBases (MSigDB) KEGG analyses. Cut-off for reporting was a false discovery rate q value of < 0.25, and normalized enrichment scores (NES) are shown, with darker colors indicating more enrichment. Open circles indicate cell cycle-related hallmarks, closed circles indicate DNA damage repair-related hallmarks. Right: Selected GSEA MSigDB Hallmarks pathways are shown with NES and false discovery rate (FDR) Indicated cell lines were treated as depicted in Figure 2. Data are depicted as mean +/- standard deviation of three independent biological experiments. Statistical significance was determine by two-tailed Student"s t test where *=p<0.05, **=p<0.01,***=p<0.001, ****=p<0.0001. LNCaP: E2F1, p=0.0159; PCNA, p=0.0217; MCM7, p=4.0936e-6; CCNA2, p=0.0005. C4-2: E2F1, p=0.0074; PCNA, p=0.1258; MCM7, p=3.7471e-5; CCNA2, p=0 ChIP-qPCR after C4-2 cells were treated as depicted in Figure 2. Data are depicted as mean +/- standard deviation of three independent biological experiments Athymic nude mice were injected with C4-2 cell mixed with matrigel. Once tumors became 100mm3, mice were treated with either vehicle control or veliparib. 72 hours later, tumors were harvested, RNA was isolated and used for qPCR quantification of the indicated transcripts. Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples, +/- standard deviation of three independent xenograft tumors Prostatectomy tissue (n=6) was cultured as previously described, and treated with either vehicle control or veliparib for six days. RNA was then harvested from the tissues and used for qPCR quantification of the indicated transcripts. Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples Each individual tissue is depicted by a separate bar color