EMBO/sd-character-level-ner huggingface dataset:
Figure 3A. Cell lysates of LN229 cells were subjected to IP using Axin antibody or control IgG, followed by IB with indicated antibodies. B. Axin siRNA or control siRNA were transfected into LN229 cells, after 36 hours, lysate of the cells was performed with co-IP using FoxM1 antibody followed by IB with indicated antibodies. C. Axin siRNA or control siRNA were transfected into LN229 cells for 36 hours. Lysates of the cells were subjected to IB with FoxM1 phospho-S474 antibody. D. Cell lysates of LN229 cells with or without Wnt-3a treatment for 4 hours were subjected to IP using Axin antibody or control IgG, followed by IB with FoxM1 and Axin antibodies.. E. Axin siRNA or control siRNA with HA-Ubiquitin were transfected into 293T cells, after 36 hours, cells were treated with MG132 with or without Wnt-3a (50 ng/ml) for six hours. Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies. F. Wild type Flag-FoxM1 or S474A mutant was transfected into 293T cells after 36 hours, cells were treated with MG132 for six hours. Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies. G. HA-Ubiquitin and/or FBXW7, GSK3β CA, Flag-FoxM1 were co-transfected into 293T cells, after 36 hours, cells were treated with MG132 for six hours. Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies. H. FBXW7 siRNA or control siRNA and HA-Ubiquitin were transfected into 293T cells, after 36 hours, cells were treated with MG132 with or without Wnt-3a (50 ng/ml) for six hours. Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies.
CBS is a prognostic biomarker for patients with IDH mutant 1p19q co-deleted gliomas.Figure 6A) Comparative gene expression analysis of key enzymes involved in GSH and cysteine synthesis and supply in IDHwt (n=220), IDHm with 1p/19q co-deletion (n=151) or IDHm without 1p/19q co-deletion (n=226) patients based on TCGA RNA-seq data from a cohort of 597 patients. CBS: Cystathionine beta synthase, key enzyme of the transsulfuration pathway. GCLC: catalytic subunit of glutamate-cysteine ligase. SLC7A11: gene encoding xCT, the cystine-glutamate antiporter. B) Kaplan-Meier curves of CBS gene expression versus overall survival in patients with IDHwt, IDHm with or without 1p/19q co-deletion. Statistical significance for the survival analysis is shown as: pv: p-value, ns= non significant.
Summary of observations in the three bacterial species investigated in this study.Figure 5Schematic representations of the various assembly and disassembly sub-complexes observed in this study in L. pneumophila, P. aeruginosa and S. oneidensis (wild type and mutant strains), respectively. The blue shaded area in (A) represents an intact IM sub-complex in the vicinity of a PL sub-complex. Note that the boundaries between the different parts in these schematics are tentative.
Figure 5A. The localization of ABH1 was analysed by immunofluorescence in HEK293 cells. ABH1 (green) localisation and mitochondria stained with a Mitotracker (red) are shown separately and in an overlay with DAPI to indicate nuclei. The scale bar represents 5 m. B. HEK293 cells expressing ABH1-HisPrcFLAG (ABH1) or the HisPrcFLAG-tag alone (FLAG) were UV crosslinked (UV) and protein-RNA complexes were affinity purified. Co-precipitated RNA was isolated and analysis by northern blot using probes against the mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. Inputs (0.1%) are shown on the left and eluates (50%) on the right.
Figure 4A. Western blot illustrating abundant Bcl11b and Gata3protein expression in Myc/Bcl2+DN2- but not LSK- or GMP-derived blasts. Thymus (Thy) and bone marrow (BM) extracts from healthy WT mice served as controls. Detection of α-tubulin or valosin-containing protein (VCP) ensured comparable loading. B,C. Proliferation assays illustrating growth of Myc/Bcl2+ DN2- (B) or GMP-derived (C) blasts after lentiviral transduction with the indicated shRNAs. The shRNA-transduced cells were flow-sorted based on co-expression of the RFP-reporter and seeded on OP9/OP9Dll1 1:1 layers. Growing cells were RFP-sorted for 3 rounds within 21 days of culture and percent growth was calculated over the initially seeded cell number in relation to the Luc control. Shown are results of 4 independent experiments with cells from 4 different DN2- and 3 different GMP-leukemic animals. Luc= control shRNA targeting luciferase (Suzuki et al., 2003). At least 2 different shRNAs were used against Bcl11b or Gata3 as indicated. * = p≤0.05, Student's t-test. D. Representative FACS plots showing CD45.2-donor splenocytes of a secondary (CD45.1) recipient 23 weeks after transplantation with 4103RFP+Myc-Bcl2+DN2-leukemia blasts carrying the indicated shRNAs. Luc: luciferase control shRNA. The data are representative of 2 Gata3, 4 Bcl11b, and 2 luciferase shRNA-transplanted animals.
Figure 5Generation of isogenic mESC lines expressing WT and arginine methylation-deficient mutant (RK) METTL14. Mettl14 KO mESCs were transfected with Flag-tagged WT or RK mutant METTL14 using a lentivirus expression system. The expression of METTL14 and METTL3 in these cells was detected by Western blot analysis using anti-METTL14 and anti-METTL3 antibodies. ACTIN was used as a loading control. m6A levels are reduced in mESCs expressing arginine methylation-deficient (RK) mutant METTL14. The mRNA purified from WT, Mettl14 KO, KO+WT, and KO+RK mESCs was subjected to LC-MS/MS analysis to quantify m6A levels (presented as the m6A/A ratio). data from three independent replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01, ***, p < 0.001. Morphology and alkaline phosphatase (AP) staining of mESCs expressing WT, Mettl14 KO, KO+WT, and KO+RK METTL14.Scale bar: 400 µM Quantification of AP-positive clones in (C). data from three independent replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01, ***, p < 0.001. Proliferation of mESCs expressing WT, Mettl14 KO, KO+WT, and KO+RK METTL14 over a 5-day period. Each point represents the average of three independent replicates and error bars represent standard deviation (SD). data from three independent replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01, ***, p < 0.001. Sequence motifs of m6A-enriched regions in KO+WT and KO+RK mESCs (upper panels). Topological distribution of normalized m6A peaks across the 5' UTR, CDS and 3' UTR of mRNAs (lower panels). Cumulative distribution of log2 m6A peak intensity in KO+WT and KO+RK mESCs. Statistical analysis was performed using the Wilcoxon test to measure the median difference of peak intensities between the two groups. Overlay of m6A distributions across the 5' UTR, CDS, and 3' UTR of mRNAs in KO+WT and KO+RK mESCs (upper panel). Statistical analysis of differential m6A peaks in KO+RK vs. KO+WT mESCs (lower panel). The y-axis represents the q-value (-log10). The dashed grey line indicates q-value = 0.05. Gene Ontology (GO) analysis of genes harboring METTL14 arginine methylation-dependent m6A sites. Statistical analysis was performed using Hypergeometric test. The p-value for the enrichment of each biological process (GO term) is shown.
Figure 3Schematic of the 4sU-DRB-seq labeling protocol. Meta-gene profile of normalized 4sU-DRB-seq reads in WT/WT and slow/slow ESCs. Density and violin plot of elongation rate (bases/min) calculated for genes common in all genotypes in WT/WT, WT/slow and slow/slow ESCs. Box
Figure 5A) Experiments shown for an exemplary group of 8 SLE patients and two healthy individuals. Dose-dependent reactivity of FcγRs IIIA and IIB/C was observed only for SLE patient sera and not for sera from healthy individuals. Each titration represents one donor measured in technical duplicates. B) Activation of FcγRs IIB/C and IIIA by patient serum is mediated by serum derived sICs. Patient serum samples were depleted of sICs by PEG precipitation and the supernatant (SN) was compared to untreated serum regarding FcγR activation (left). Shown is one exemplary experiment performed in technical duplicates. Error bars indicate reproducibility as range. IgG concentration in the precipitate (PC), supernatant (SN) or unfractionated serum respectively is shown in the bar graph (right). C-D) Correlation of FcγR activation data from A with (C) patient SLEDAI or (D) conventional SLE disease markers (α-dsDNA levels indicated as IU/ml or C3d concentrations indicated as mg/L). Left panel: FcγRIIIA; right panel: FcγRIIB/C. FcγR activation from a dose-response curve as in A was calculated as area under curve (AUC) for each SLE patient represented by individual dots. AUC from SLE patients are expressed as fold change compared to the healthy control mean (n=4). SLE patients with α-dsDNA levels below 50 IU/ml and C3d values below 6 mg/L were excluded. One-tailed Spearman's. Dotted lines show 95% confidence bands.
Figure 3Figure 3. RPA recruits SLFN11 to sites of DNA damage. (A-C) SF268 cells stably expressing Flag-tagged SLFN11 were infected with non-target, or RPA1-specific lentiviral shRNAs. 48 hr after infection, cells were treated with CPT (1 µM). 3 hr later, cells were subjected to immunostaining using anti-RPA2 and anti-Flag antibodies. Representative SLFN11 and RPA2 foci were shown (A). . Scale bar, 10 µm. Quantification of RPA2 and SLFN11 foci formation using NIH Image J software (B) Error bars are s.d.; n=3. Knock-down efficiency of RPA1 in SF268 cells was confirmed by immunoblotting (C). (D) RPA1 is required for the accumulation of SLFN11 at laser-induced DNA damage tracks. A SF268 cell line stably expressing wild-type RPA1 under the control of a tetracycline-inducible promoter was generated. The resulting cell line infected with RPA1-specific lentiviral shRNA targeting the 3'-UTR of RPA1 transcript was induced by doxycycline addition for 48 hr and then laser-microirradiated. Thirty minutes later, cells were fixed and stained with anti-RPA2 and anti-SLFN11 antibodies. Scale bar, 10 µm. (E) The C terminus of SLFN11 is required for its foci formation. SF268 cells were transfected with plasmids encoding Flag-tagged wild-type or mutant of SLFN11. Immunostaining experiments were performed 3 hr after CPT (1 µM) treatment using indicated antibodies. Scale bar, 10 µm. (F-H) SF268 cells stably expressing Flag-tagged SLFN11 were transfected twice with indicated siRNAs. 48 hr after second transfection, cells were treated with CPT (1 µM). 3 hr later, cells were subjected to immunostaining using anti-RPA2 and anti-Flag antibodies. Representative SLFN11 and RPA2 foci were shown (F). Scale bar, 10 µm. Quantification of RPA2 and SLFN11 foci formation using NIH Image J software (G). Error bars are s.d.; n=3. Knock-down efficiency of Mre11 and CtIP in SF268 cells was confirmed by immunoblotting (H).
,4-DC induced TFEB and TFE3-depdendent autophagy (A-F) U2OS-GFP-LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB (siTFEB-#1, siTFEB-#2, siTFEB-#3) or scrambled siRNA (siCtr) (A-C), or U2OS wild type (wt) and TFEB knockout (TFEB KO) GFP-LC3 cells (D-F) were treated with 3,4-DC as indicated for 16 h.Figure 5U2OS-GFP-LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB (siTFEB-#1, siTFEB-#2, siTFEB-#3) or scrambled siRNA (siCtr) were treated with 3,4-DC as indicated for 16 h. TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS-PAGE and immunoblot U2OS-GFP-LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB (siTFEB-#1, siTFEB-#2, siTFEB-#3) or scrambled siRNA (siCtr) were treated with 3,4-DC as indicated for 16 h. GFP-LC3 dots were quantified as indicator of autophagy Data are means ± SD (* = p < 0.05, ** = p < 0.01 versus siCtr or WT cells treated with 3,4-DC). Representative images are shown Scale bar equals 10 µm. U2OS wild type (wt) and TFEB knockout (TFEB KO) GFP-LC3 cells were treated with 3,4-DC as indicated for 16 h. TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS-PAGE and immunoblot U2OS wild type (wt) and TFEB knockout (TFEB KO) GFP-LC3 cells were treated with 3,4-DC as indicated for 16 h. GFP-LC3 dots were quantified as indicator of autophagy ata are means ± SD (* = p < 0.05, ** = p < 0.01 versus siCtr or WT cells treated with 3,4-DC). Representative images are shown cale bar equals 10 µm. U2OS wild type (WT), TFEB or TFE3 knockout, or double knockout (TF DKO) cells were treated with 30 µM 3,4-DC for 16 h. Following, cells were collected and SDS-PAGE and immunoblots were performed as described before. TFEB, TFE3, LC3, p62, and GAPDH protein levels were measured with specific antibodies (G). U2OS wild type (WT), TFEB or TFE3 knockout, or double knockout (TF DKO) cells were treated with 30 µM 3,4-DC for 16 h. GFP-LC3 dots were quantified as indicator of autophagy (H). Data are means ± SD (* = p < 0.05, ** = p < 0.01, *** = p < 0.001 versus Ctr, respectively). (I) U2OS WT, knockout for TFEB (TFEB-/-) or TFE3 (TFE3-/-), double knockout (DKO) were treated with 3,4-DC for 24 h and stained with LysoTracker Red for 30 min. Thereafter, the red (positive) dots were measured. Data are means ± SD (* = p < 0.05; ** = p < 0.01; *** = p < 0.001). (J) Relative mRNA expression levels of key autophagy genes such as Lamp1, Lc3b and p62 were detected in controls and 3,4-DC treated U2OS cells. Data is depicted as a heatmap showing means of at least three independent experiments (*** = p < 0.001 versus WT/Ctr and # = p < 0.05, ## = p < 0.01, as compared to WT treated with 3,4-DC).
Figure 5Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (A) Representative results for individual MTB-reactive plasmablast antibodies. Mean and SEM are indicated. Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (B) Direct comparison of all tested MTB-reactive IgA (n=12) and IgG (n=26) antibodies. Mean and SEM are indicated. Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (C) Representative results for individual HBHA-reactive memory B cell antibodies from TB patients and HCW. Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (D) Same as in (B) but with HBHA-reactive antibodies as shown in (C). (D) Direct comparison all tested HBHA-reactive IgA (n=6) and IgG (n=7) antibodies. Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (E) Direct comparison of the indicated MTB-reactive antibodies expressed as IgA and IgG. Mean and SEM are indicated. P-values were determined using non-parameric t-test, * p<0.1; **p<0.01, ns: p>0.5.
Figure 5WT and Mecp2 null MEF cells were starved for 48 h and then treated with the HDAC6 inhibitor tubacin (TUB) (1 μM) for 48 h or with DMSO (VEH) or left untreated (UT). Cells were fixed, stained for anti-acetylated α-tubulin and cilia were counted. The graph shows the percentage of ciliated cells out of three independent experiments (mean ± S.E., *p<0.05, **p<0.01, by two-way ANOVA followed by Bonferroni post hoc test). Two-way ANOVA reported a significant genotype effect (F(1, 10) = 17.94; p<0.01), a significant tubacin treatment (F(2, 10) = 4.425; p<0.05) and a significant interaction (F(2, 10) = 7.266; p<0.05). Primary cortical neurons were treated at DIV5 with DMSO (VEH) or tubacin (TUB, 1 μM) for 48 h, or left untreated (UT), fixed at DIV7 and stained with anti-AC3 antibody. The graph shows the percentage of ciliated cells obtained from three independent experiments counting n=300 UT cells, n=216 VEH treated cells, n=267 TUB treated cells for each genotype (mean ± S.E., *p<0.05, **p<0.01, by two-way ANOVA, followed by Bonferroni post hoc test). Two-way ANOVA reported a significant genotype effect (F (1, 15) = 23.42; p<0.001), a significant treatment effect (F (2, 15) = 11.25; p<0.01) and a significant interaction (F (2, 15) = 4.853; p<0.05). MECP2 silenced or control hTERT-RPE-1 cells were starved for 48 h, treated with tubacin (TUB, 1 µM) for 24 h and then exposed to 200 nM SAG for 24 h in the presence of tubacin. Cells were stained with anti-Smo and anti-acetylated α-tubulin antibodies and the percentages of Smo-positive cilia were calculated. The graph shows the averages of three independent experiments (mean ± S.E., **p<0.01, by three-way ANOVA followed by Tukey's post hoc test). Statistical analysis reported a significant difference between genotype (F (1, 2) = 21.47; p<0.001), a significant SAG effect (F (2, 2) = 154.5; p<0.0001) and a significant tubacin effect (F (1, 2) = 25.18; p<0.001). In details, a significant interaction between genotype and SAG treatment (F (2, 2) = 5.413; p<0.05) and between SAG and tubacin treatment (F (2, 2) = 6.939; p<0.01) were reported, demonstrating respectively a difference between CTRL and MeCP2 depleted cells in response to SAG and a different SAG effect when cells were treated with tubacin. Representative immunostaining of Smo (green) and acetylated α-tubulin (red) in control (CTRL) and silenced (siMeCP2) hTERT-RPE-1 cells, after stimulation with 200 nM SAG in the presence or absence of the HDAC6 inhibitor tubacin (TUB). Scale bar 50 µm, and 20 µm in the enlarged image.
Addition of corticosteroids affects biofilm formation and antibiotic tolerance of H.Figure 4Addition of corticosteroids affects biofilm formation and antibiotic tolerance of H. influenzaeH. influenzae biofilms were developed after 24 h in μ-well chambers in sBHI medium with and without corticosteroid and were then treated with the antibiotic azithromycin as specified.(i) H. influenzae wild-type with DMSO (solvent control); (ii) H. influenzae wild-type treated with 1 μM beclomethasone (BEC); (iii) H. influenzae wild-type treated with 150 μg/ml azithromycin (AZO); and (iv) H. influenzae wild-type treated with 1 μM beclomethasone (BEC) and 150 μg/ml azithromycin (AZO). For these experiments, H. influenzae was visualized with SYTO9 (green strain), as described in Materials and Methods. Scale bars = 20 μm. Images shown are representative of 12 images from five independent experiments.The biofilm biomass after treatments was quantified using COMSTAT. Data are presented as the average of five replicates, with error bar representing the standard deviation of the data. Statistical significance by two-tailed Student's t-test is indicated: **P < 0.01.
IAHCs have a monoclonal, proliferative origin but become polyclonal at later stage.Figure 2Schematic representation of time of induction of recombination in VeCadCreER:Confetti pregnant females and analysis of embryos. Tamoxifen was injected before cluster initiation (E7.5 or E8.5) and embryos collected at the peak of cluster formation (E10.5 or E11.5). Quantification of recombination efficiency at E10.5 and E11.5 using confocal images. Bars represents the mean±SD of the percentage of cells expressing single colored cassettes in CD31+ endothelial cells (n=2). Transversal section of a recombined VeCadCreER:Confetti AGM from an E10.5 embryo and immunostained with CD31 (C) (white in image, black in label) Multistack reconstruction of confocal images. Scale bars 30μm Transversal section of a recombined VeCadCreER:Confetti AGM from an E10.5 embryo and immunostained with Kit (D) (white in image, black in label). Multistack reconstruction of confocal images. Scale bars 20 μm Schematic representation of possible outcomes in the IAHC after recombination: Clusters derived from sequential division of one colored cell will be unicolored (left panel). Clusters will be multicolored (right panel) when derived from several, independently recombined endothelial cells. Analyses of recombination in 40 clusters. Bars representing single clusters are grouped based on the number of Kit+ cells. Recombination induction follows the schema in the black square. (n=12 embryo). Representative confetti recombination on clusters of different sizes. Small cluster are monoclonal (left panel), while larger clusters are polyclonal (right panel). Inlet shows relevant confetti channel. Kit is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Scale bars 5 μm.
Xenograft of CTC-ITB-01 in immunodeficient mice confirms tumorigenic and metastatic potential: A In vivo bioluminescence imaging of CTC-ITB-01 in four NSG mice.Figure 6A In vivo bioluminescence imaging of CTC-ITB-01 in four NSG mice. Blue indicates low radiance, red indicates high radiance. B Logarithmic plotting of in vivo radiance increase over time. Curves represent means ± SEM of measurements performed on multiple glands (n=8). C H&E staining of xenografted mouse mammary glands show formation of primary tumors. D Stereoscope image of a mammary gland of immunodeficient NSG mouse. Fluorescently tagged CTC-ITB-01 cells in green. E IHC staining for ER, confirms retained in vivo ER-positivity of formed tumors. Representative images of ER staining in higher (left panel) and lower (right panel) magnification of 2 different areas of a xenografted mammary gland. F Representative ex vivo luminescence images of indicated organs from engrafted mice. Organs include brain (1), lung (2), liver (3) and bones (4). G Scatter blot showing ex vivo radiance intensity of metastatic organs in three different mice (M1, M2, M3), indicating highest intensity in liver and bone. Data represent means ± SD of measurements performed on metastatic organs derived from three different mice.
Schistosomal-EVs are internalized by Th cells.Figure 3EVs were purified from either schistosomal-growing medium (supernatant) or unused schistosomal-medium as control. Both pellets were stained using Thiazole Orange. Purified labeled ~5 × 106 EVs were incubated with 5 × 105 freshly purified Th cells that were stimulated with anti-CD3 and anti-CD28 antibodies for 10 minutes. Images from the same slide were taken by inverted confocal microscopy at the indicated time points. The arrows in the enlarged images point toward cells labeled with EVs. The scale bars in all images represent 20μm. The percentage of labeled cells was calculated by dividing the number of labeled cells by the total numbers of cells in the same image. The mean +/- SEM was calculated from 3 independent images
Overview of the workflow.Figure 1Major steps involved when quantifying transcription (RNA-seq) and translation (Ribo-seq) and the additional cellular features measured. Elements required for quantification in absolute units are highlighted in red. Model for calculating the translation initiation rate of a ribosome binding site (Eq. 2). Model for calculating translation termination efficiency of a stop codon (Eq. 3). Star denotes the location of the stop codon. Model for calculating translational frameshifting efficiency between two coding regions 'A' and 'B' in zero and -1 reading frames, respectively (Eq. 4).