EMBO/sd-character-level-ner huggingface dataset:
T.Figure 1A, Primary HBMEC were challenged with type I (RH) or type II (ME49) strains of T. gondii for the indicated time points. Cell lysates were used to probe for total Akt and phospho-Ser473 Akt by immunoblot. B, C, Mouse endothelial cells (mHEVc; B) and human retinal pigment epithelial cells (RPE, C) were challenged with RH T. gondii followed by assessment of Akt phosphorylation. B, C, Mouse endothelial cells (mHEVc; B) and human retinal pigment epithelial cells (RPE, C) were challenged with RH T. gondii followed by assessment of Akt phosphorylation. D, HBMEC were challenged with either live or killed T. gondii. Cell lysates were subject to immunoblotting as indicated. E, HBMEC were transiently transfected with a plasmid encoding PH-Akt-GFP. Cells treated with or without LY294002 (20 µM) for 1 h prior to challenge with T. gondii-RFP. Cells were examined by immunofluorescence at 5 min post-challenge to examine accumulation of PH-Akt-GFP around the parasites. F, HBMEC were incubated with LY294002 or vehicle for 1 h prior to challenge with T. gondii. Densitometry data represent means ± SEM of 4 experiments. A vertical line was inserted between densitometry data from control and LY294002-treated cells to indicate that band densities from infected cells treated with or without LY294002 are compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. G, HBMEC were transiently transfected with control siRNA or PI3K p110α siRNA. Cells expressing either control siRNA or PI3K p110α siRNA were challenged with T. gondii 48 h after transfection. Cell lysates were subject to immunoblotting as indicated. Densitometry data are shown as above and represent means ± SEM of 3 experiments. Results shown are representative of 3-4 independent experiments.
Figure 3Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). 24-hours later, cells were incubated with (A) TRITC-dextran (70 kDa, shown as red signal) Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as "CoLoc." Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker. Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). 24-hours later, cells were incubated with (B) DQ-ovalbumin, for 30 min before washing and fixation. Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as "CoLoc." Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker. Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). (C, D) Cells were further stained for (C) EEA1, or (D) Lamp1, which was detected with Cy5 dye (show as red signal). Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as "CoLoc." Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker. E) 25 cells across three independent experiments were analyzed for each condition. % of Rab10 vesicles positive with the indicated marker in each cell were quantified. Each dot represents the mean results from vesicles analyze in one cell. The group means of % Rab10 vesicles positive with the marker are shown, with error bars representing ± SEM. Significance was assessed by Mann-Whitney test for each marker, where ** representing p<0.01, **** p<0.0001 and N.S p>0.05.
Figure 5(A) Schematic representation of the potential miR-17 (blue) and miR-92a (green) binding sites in the 3'UTR of the BCL11B gene. 3 nucleotides in the 3 putative miR-17 binding sites which are targeted for mutagenesis, used in a 3'UTR luciferase reporter assay described in B, are depicted in red. (B) Transfection of miR-17 Pre-miR miRNA Precursor, but not of miR-92a, specifically targets the wild-type (black bars) 3'UTR of BCL11B in a 3'UTR luciferase reporter assay following cotransfection of HEKT cells in comparison to the controls (transfection of no miRNA or a control miRNA). Mutation of the predicted miR-17 binding sites in the 3'UTR BCL11B reporter construct prevents miR-17 dependent targeting (grey bars). Graphs show mean firefly luciferase values (from 3'UTR reporter constructs) following normalization to the cotransfected control Renilla luciferase and relative to control sample without miRNA (n=5 independent experiments, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a paired t-test). (C) Quantitative RT-PCR gene expression analysis of BCL11B in control (black bar) or miR-17 (grey bar) transduced CD34+ thymocytes after 24h of culture on coated Fc-DLL1 plates, relative to the geometric mean of ACTIN and GAPDH and relative to the control-transduced sample (n=3 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test). (D) Quantitative real-time RT-PCR gene expression analysis of BCL11B in control (black bar) or miR-17 sponge (white bar) transduced CD34+ thymocytes, relative to the geometric mean of ACTIN and GAPDH (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).
Figure 2(a) Deposition of uPA or PAI-1 (green) in the cremaster muscle of WT mice as assessed separately ex vivo by confocal laser scanning microscopy 6 h after intrascrotal injection of TNF or saline, PECAM-1/CD31+ postcapillary venules (red) and Ly-6G+ neutrophils (purple) are depicted. Representative images are shown (scale bar: 50 µm). (b) Expression of PECAM-1/CD31 (red) and ICAM-1/CD54 or VCAM-1/CD106 (green) in the cremaster muscle of WT mice as assessed ex vivo by confocal laser scanning microscopy 6 h after intrascrotal injection of recombinant murine uPA-PAI-1 or saline, representative images (scale bar: 50 µm) and quantitative data are shown (mean±SEM for n=4 mice per group; #p<0.05 vs. saline; t test). (c) Production of TNF was analyzed in mouse RAW 264.7 macrophages bEnd.3 microvascular endothelial cells as assessed in vitro by multi-channel flow cytometry upon exposure to recombinant murine uPA-PAI-1, TNF, or saline, quantitative data are shown (mean±SEM for n=4 experiments per group; #p<0.05 vs. saline; One-way ANOVA / t test). (d) surface expression of ICAM-1/CD54, VCAM-1/CD106, uPA, and PAI-1 on mouse bEnd.3 microvascular endothelial cells as assessed in vitro by multi-channel flow cytometry upon exposure to recombinant murine uPA-PAI-1, TNF, or saline, quantitative data are shown (mean±SEM for n=4 experiments per group; #p<0.05 vs. saline; One-way ANOVA / t test). (e) Binding of ICAM-1/CD54-Fc or VCAM-1/CD106-Fc to primary mouse neutrophils ('mAB 24' recognizing high-affinity conformation, 'Kim127' recognizing intermediate and high affinity conformation of β2 integrins) as assessed upon exposure to recombinant murine/human uPA-PAI-1, TNF, or saline by multi-channel flow cytometry, a representative histogram plot and quantitative data are shown (mean±SEM for n=4-7 mice/human blood samples per group; #p<0.05 vs. saline; One-way ANOVA). (f) binding of conformation-specific mABs to primary human neutrophils ('mAB 24' recognizing high-affinity conformation, 'Kim127' recognizing intermediate and high affinity conformation of β2 integrins) as assessed upon exposure to recombinant murine/human uPA-PAI-1, TNF, or saline by multi-channel flow cytometry, a representative histogram plot and quantitative data are shown (mean±SEM for n=4-7 mice/human blood samples per group; #p<0.05 vs. saline; One-way ANOVA). (g) Quantitative analysis of LFA-1/CD11a clustering on the surface of mouse neutrophils under increasing flow conditions in vitro in the presence of either E-selectin/CD62E + ICAM-1/CD54, E-selectin/CD62E + ICAM-1/CD54 + KC, or E-selectin/CD62E + ICAM-1/CD54 + KC + uPA-PAI-1 as assessed by spinning disk confocal microscopy, representative images and quantitative data are shown (scale bar: 10 μm; mean±SD; n=3 mice; n=10-13 flow chambers, n =122-222 cells); *p<0.05 vs. E-selectin/CD62E + ICAM-1/CD54; One-way ANOVA).
Figure 6(A) Intra-architectural mutations: both change of motif strength and motif position within the same construct. The Mec2 motif composition is shown here as an example. (B) Linear regression applied to predict the synthetic promoter activity based on individual motif features (intra-architectural mutations). The measured expressions (on the y-axis) for 6 tested core promoter sequences with combinatorial motif mutations compared to the predicted expressions (on the x-axis) from the linear regression (log2 scale). Red solid line: y = x; red dashed lines: y = x ± 3×SD, where SD denotes the median of all standard deviations over all measured synthetic promoter constructs. It is an estimate for the noise in the expression measurements. The linear regression model can explain on average 88% of the variance in expression (average r = 0.88). (C) Context exchange between different core promoters: the motifs of promoter 2 with their respective relative distance are conserved, and are incorporated in the sequence context of promoter 1. (D) Effect of motif context sequence exchange. Heatmap depicting the mean expression fold changes caused by motifs (y-axis) inserting of RpL5, RpL36A, thoc6, CG8157, and cas to different context sequences (x-axis). The heatmap shows the expression changes relative to wild-type expressions of the context-origin promoters CG10915, CG15674, cas, CG8157, thoc6, RpL36A', and RpL5, respectively. (E) Inter-architectural mutations: block-wise combinatorial mutations between different core promoters. The motifs together with their sequence context within a block are swapped with others. (F) Linear regression analysis for inter-architectural block-wise combinatorial mutations. The measured expressions (on the y-axis) for inter-architectural block-wise combinatorial mutations compared to the predicted expressions (on the x-axis) from the linear regression fit (log2 scale). Red solid line: y = x; red dashed lines: y = x ± 3×SD, where SD denotes the median of all standard deviations over all measured synthetic promoter constructs. Pearson coefficient 0.81.
Figure 2A Maximum intensity projections of smFISH fluorescence microscopy z-stacks of Jurkat T cells stained for the indicated genes after 1-hour (Nfkbia, Tnfaip3, Tnf, Il8) or 4-hour (Il6, Csf2) treatment with 20 ng/mL TNF. Nfkbia, Tnfaip3, Tnf, and Il6 were labeled with Quasar 670, and Il8 and Csf2 were labeled with fluorescein. All images were filtered as described in Materials and Methods. Brightness and contrast were enhanced for visualization. Scale bars: 10 μm. B Bar graphs of mean of smFISH distributions before and after TNF treatment for the indicated genes. Cells were combined from three replicates (Nfkbia 1h; Tnfaip3 1h; Tnf 1h, 2h; and Il6 2h, 4h) two replicates (Nfkbia 2h) or one replicate (Tnfaip3 2h; Il8 1h, 2h; Csf2 2h, 4h). Basal data are same as in Fig. 1E. Error bars indicate bootstrapped 95% CIs for the samples in (C). Significant differences indicated by non-overlapping CIs. C C Probability density plots of single-cell mRNA distributions from smFISH as described in (B) before and after treatment with 20 ng/mL TNF for the indicated time points. D-E Graph of log10(mean) versus log10(CV2) for endogenous gene targets that maintain CV2 (D) or decrease CV2 (E) after treatment with 20ng/mL TNF in Jurkat T cells (black), HeLa cells (red) or murine bone marrow derived macrophages (green). Gray shading indicates 95% CI of basal trendline. Poisson trendline indicated by dashed line.
Monoclonal and polyclonal anti-AdV antibodies trigger inflammasome activation in human macrophages (A) THP-1 cells were stimulated with (50 000 particles/cell) AdV-GFP and infection after 24 h measured by flow cytometry.Figure 1THP-1 cells were stimulated with (50 000 particles/cell) AdV-GFP and infection after 24 h measured by flow cytometry. Cells were stimulated as in (A) with AdV-GFP or AdV-GFP complexed with 20 μg/ml h9C12 or 20 mg/ml human IVIg. Cell supernatants were harvested at indicated time-points and cytokines measured by ELISA (B) Cells were stimulated as in (A) with AdV-GFP or AdV-GFP complexed with 20 μg/ml h9C12 or 20 mg/ml human IVIg. pro- and cleaved IL-1β was measured in whole cell lysates or in supernatants by western blot (C). HMDM were primed with 10 μg/ml pI:C for 2h, then stimulated for 16h with virus +/- antibody. Cytokines in the supernatants were measured by ELISA.
Figure 4(A) In vitro cardiac differentiation protocol with lactate enrichment (from days 14 to 16 after induction) performed in seven isogenic MitoA-derived hiPSCs with different mtDNA genotypes. (B) mtDNA NGS showing the position and mutation load of GPMs in seven isogenic MitoA-derived hiPSCs clones and their differentiated counterparts. The mtDNA genotype is stable during in vitro cardiac differentiation and lactate enrichment. MitoA hiPSCs 57 and 65 (wild-type) are labelled in black, MitoA hiPSCs 61 and 69 (m.3243A>G mutant) are labelled in red and MitoA hiPSCs 58, 59 and 217 (with amplified GPMs in nuclear reprogramming) are labelled in blue. (C) Quantification of the percentage of mature hiPSCs-derived cardiomyocytes in direct differentiation of hiPSC clones. Representative histograms showing the percentage of the cells expressing mature cardiac markers: myosin heavy chain (MF20), troponin T (cTnT) and troponin I (cTnI) by flow cytometry, in MitoA-57-derived cardiomyocytes (wild-type) and MitoA-69-derived cardiomyocytes (83% m.3243A>G). For complete quantification of cardiac markers in all hiPSCs-derived cardiomyocytes generated. (D) Immunofluorescence images of cardiac markers (cTnT and MF20) showing similar sarcomere structures in hiPSC-derived cardiomyocytes wild-type (MitoA-57) and mutant (MitoA-69). (E) Oxygen consumption rate (OCR) in hiPSCs. OCR reported as a % of that in MitoA-57 hiPSC, the control line. MitoA-57 and MitoA-65 hiPSCs wild-type (in black), MitoA-61 and MitoA-69 m.3243A>G mutant (in red), MitoA-58, MitoA-59 and MitoA-217 hiPSCs amplified GPMs (in blue). Error bars are mean ± SD and OCR data are representative 3 biological replicates in all samples.(F) Oxygen consumption rate in hiPSCs-derived cardiomyocytes at day 19 of cardiac differentiation. OCR reported as % of that in MitoA-57-derived cardiomyocytes, the control line. MitoA-57 and MitoA-65 derived cardiomyocytes wild-type (in black), MitoA-61 and MitoA-69 derived cardiomyocytes mutants m.3243A>G (in red), MitoA-58, MitoA-59 and MitoA-217 derived cardiomyocytes with amplified GPMs (in blue). Error bars are mean ± SD and OCR data are representative of two independent in vitro cardiac differentiation experiments, with at least 3 biological replicates in each sample, and significance established using Student's t-test.
a) Relative acetylation of histone H3 Lys 9 and 14 residues determined by quantification of immunoblot analysis performed at day 20 of the ageing experiment shown in panel b.figf5(a) Relative acetylation of histone H3 Lys 9 and 14 residues determined by quantification of immunoblot analysis performed at day 20 of the ageing experiment shown in panel b. Data represent means ± s.e.m. (n = 3; *P 0.05). (b) Chronological ageing of wild-type (▪, o) and Δiki3Δsas3 (▴, ▿) with (open symbols) or without (closed symbols) addition of spermidine (4 mM) at day 1. Data represent means ± s.e.m. (n = 4). (c) Quantification (FACS analysis) of phosphatidylserine externalization and loss of membrane integrity (annexinV/PI costaining) as well as ROS production (DHE->Eth conversion) performed at day 20 of the experiment shown in b. Data represent means ± s.e.m. (n = 4; *P 0.001). (d) Relative inhibition of histone acetyltransferase activity (HAT-activity) by spermidine determined by an in vitro HAT-activity assay of yeast nuclear extracts of wild-type cells. Data represent means ± s.e.m. of three independent experiments (*P = 0.024). (e, f) Transcriptional change (obtained by Affymetrix array) by spermidine treatment of all autophagy-related genes categorized into GO terms of macroautophagy (GO:0016236), microautophagy (GO:0016237) or autophagy in general (GO:0006914) at day 3 (e) and day 10 (f) of chronological ageing yeast. Data represent means of two independent arrays from independent biological samples. (g) Relative change of ATG7, ATG11 and ATG15 mRNA levels by spermidine supplementation (normalised to controls) after ten days of chronological ageing as determined by reverse transcriptase real-time PCR. Data represent means ± s.e.m. (n = 3; *P 0.05 and **P 0.01). (h) Relative histone H3 Lys 18 acetylation of indicated promoter regions normalized to the acetylation of ATG7 promoter (pATG7) determined by chromatin immunoprecipitation with H3-K18Ac specific antibody and subsequent quantification of precipitated promoter DNA using quantitative real time PCR. Signals specific for indicated promoter regions were initially compared with non-immunoprecipitated samples and afterwards normalized to the pATG7 signals. Data represent means ± s.e.m. (n = 3). The P value was calculated using a two-factor ANOVA with promoter and condition (treated and non-treated) as independent factors.
Slug inhibition reduces PH severity in our rat model of PF-PH.Figure 7(A) Experimental schematic of the prevention protocol. All rats received Bleomycin at day 0 and MCT at day 14. Starting at day 17, rats received five intratracheal nebulizations (twice a week for three weeks) of either a small interfering RNA (siRNA) directed against Slug (Si-Slug) or scramble siRNA (Si-Scrm). Relative expression of Slug protein (B) normalized to GAPDH. Relative expression of Slug mRNA (C) normalized to GAPDH. (D) right ventricular systolic pressure (RVSP) and (E) right ventricular hypertrophy index in PF-PH rats treated with Si-Slug compared to Si-Scrm. Relative expression of fibronectin (F), Collagen I (G) and Collagen III (H) normalized to GAPDH. (I) Masson's trichrome representative images and quantification of the Ashcroft score. (J) Masson's trichrome representative images and quantification of vascular wall thickness. (K) Masson's trichrome representative images and quantification of the vascular remodeling between non-fibrotic and fibrotic areas of the lung. (L) Representative images and quantification of Ki67-positive (red) pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC, aSMA in white). The number of samples per group for each experiment are included within each bar graph.
MAPAs contain layered structures and mitochondrial proteins.Figure 5csCLEM analysis of cells transfected with GFP-p62 and treated with MG132. Red arrowheads show the dark red fluorescent beads used to correlate the images from light microscopy (LM) and transmission electron microscopy (TEM). The blue arrowhead shows a MAPA observed under LM and TEM. TEM analysis of HeLa cells treated with DMSO or MG132. Note the layered structures (red arrowheads) in the mitochondrial matrix (ii, iii), budding from mitochondria (iv), attached to mitochondria (v-vi), away from mitochondria (vii) or inside the MAPAs (viii, ix) in MG132-treated cells. Scale bar=500 nm. M, mitochondrion. N, nucleus. Immunoblot analysis of the TX-100-soluble and -insoluble fractions of HeLa cells treated with MG132 as indicated. Immunoblot analysis of the TX-100-soluble and -insoluble fractions of HeLa cells transfected with Ub-R-GFP and treated with MG132 as indicated.
Fig.Figure 6Our data indicate a two-pronged effect of PP2A on pruning: firstly, PP2A is required for timely expression of Mical via an unknown mechanism. Secondly, PP2A appears to affect actin dynamics in neurons during both the larval and pupal stages.
Figure 3A. Tumor mass volume was measured at specific time-points until the day of sacrifice (day 39 post-injection for control, day 46 and 56 p.i. for MCU-/- cl.1 and cl.2, respectively). P-values: ***p=0.0001, ***p<0.0001. B. left: in vivo metastasis at the homolateral axillary area of three representative mice per group at the time of sacrifice. Right: total flux analysis. P-values: **p=0.01, *p=0.02. C. Lymph nodes weight at the time of sacrifice. P-values: ***p=0.0010, **p=0.0014. D. Human cytokeratin 7 (CK7) IHC staining of three representative lymph nodes per group. Scale bar= 500 µm. E. Left: images of three representative lungs per group collected ex vivo at the time of sacrifice. Right: total flux analysis. P-values: **p=0.0031, ***p= 0.0004.
Figure 2Illustration of supported cell membrane sheet (SCMS) assay. Representative confocal images of SCMS expressing Tac-YFP-DATC24, incubated with pre-complexed fluorescently labelled PICK1 and unlabelled peptides as indicated (10 nM), scale bar 10 µm. Full concentration curves shown in Fig S4 and include the representative images shown here.   SCMS derived competitive binding curves for peptides as indicated, red line indicates 10 nM as shown in representative images.   Experimental outline of pre-incubation with peptide. Representative confocal images of SCMS expressing Tac-YFP-DATC24, incubated with fluorescently labelled PICK1 and subsequently unlabelled peptide as indicated (10µM). Scale bar 10 µm. Full concentration curves shown in Fig EV2 and include the representative images shown here. Efficacy of the peptides as indicated to facilitate dissociation of PICK1 from SCMS, red line indicates 10 µM as shown in representative images. Experimental outline post-incubation with peptide.