EMBO/sd-character-level-ner huggingface dataset:
Fig.Figure 3(A) Western blot analysis showing efficient knock-down of uSTAT5 in HPC7 cells on day 3 after infection. (B) RNA microarray analysis following uSTAT5 depletion by shRNA_7 in HPC7 cells grown in SCF. The 40 most upregulated genes following uSTAT5 knock-down are illustrated. Genes upregulated during megakaryopoiesis are indicated in red. (C) Gene set enrichment analysis showing significant enrichment of a megakaryocyte progenitor (MKP, Lin-Sca1-Kit+Itga2b+Slamf1+) signature in STAT5 knockdown cells. CLP, common lymphoid progenitor (Lin-Sca1lowKitlowIL7r+Flk2+); preGM, pre-grenulocyte/macrophage progenitors (Lin-Sca1-Kit+Itga2b-FcgRlowSlamf1-Englow); NES, normalized enrichment score. (D) Quantitative RT-PCR confirmed upregulation of uSTAT5 target genes following uSTAT5 depletion. Relative RNA levels were normalized to 18S RNA. Bar graphs represent means ± SD of three independent experiments. (E-F) Kinetics of the upregulation of uSTAT5 (E) and pSTAT5 (F) target genes following TPO stimulation. Each data point represents a mean ± SD. n=3; *, significant difference from time 0 (p<0.05); *, p<0.05; **, p<0.01; ***, p<0.001.
Tap-1 belongs to the superfamily of DUF4123 proteins.Figure 1Tap-1 belongs to the superfamily of DUF4123 proteinsGraphical depiction of the T6SS auxiliary gene cluster 1 of Vibrio cholerae strain N16961. Gene names are indicated above each gene. Tap-1 (yellow) is outlined in bold, and its locus tag is indicated below the gene.Graphical depiction of Tap-1 indicating the domain with homology to DUF4123.Graphical depiction of DUF4123-containing gene clusters drawn to scale. The bacterial species encoding the genes are indicated on the left. All genes encoding proteins of the DUF4123 superfamily (yellow) are outlined in bold and labeled with their locus tag.Data information: A legend for color coding is shown below (C). Legend indicates function predicted by BLAST. Genes of unknown function are colored in gray. Genes outside the operons are colored in white.
Phospholipid accumulation in Δatg5 promastigotes.Figure 6Phospholipid accumulation in Δatg5 promastigotes.Negative ion survey scans (650-900 m/z) of WT (A) and Δatg5 (B) promastigotes extracted for lipids and analysed by ES-MS, as described in Materials and Methods. a = (alkylacyl).
Figure 4Dose-dependent TFL457 effects. Cultures incubated with TMyc o TFL457 (5, 15 o 25 μM, 30 min) were treated with NMDA (2 h) and compared to cells without peptide or NMDA. Time-course TFL457 effects. Cells preincubated with peptides (25 μM, 30 min) were treated with NMDA (0-6 h). Quantitation of peptide effects. Normalized protein levels are presented relative to values obtained in cells incubated with the same peptide but no NMDA. Means ± SEM are given. Results were analyzed by a two-way ANOVA test followed by post-hoc Bonferroni test, comparing TMyc or TFL457-treated cells for each time-point (TrkB-FL, ***p = 0.0009 and *p = 0.02; CREB, *p = 0.0149 and *p = 0.0148; pCREB, ***p = 0.0007 and **p = 0.0012; MEF2D, **p = 0.0023 and **p = 0.0047. All values are respectively for 2 or 4 h; n = 6).
Figure 3A) PALB2 cell lines transfected with PALB2 siRNA followed by inducible expression of siRNA resistant PALB2 versions were exposed to 15 Gy of IR and fixed for IF 2 hours later. Z-stack images were acquired with a 60X oil objective (Deltavision). Z stack max intensity projection, background subtraction, and foci count was done using ImageJ/Fiji. (* p<0.0001, unpaired Student's t-test). Scale bar = 5μm. B) PALB2 cell lines were transfected and the exogenous PALB2 induced as in Figure 3A before they were pulsed with 10 μM EdU for 20 minutes prior to addition of 2 mM HU. 24 hours later the cells were fixed and processed for IF as in Figure 3A. Cells in S-phase (EdU+) at the time of HU treatment were Click-IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ/Fiji. (* p<0.0001, unpaired Student's t-test). Scale bar=5μm. C) HeLa DR-GFP cell line was used to analyze homologous recombination activity. Cells were transfected with PALB2 siRNA followed by transfection of I-SceI together with empty vector (EV), WT-, TMA-, or TMD-PALB2 the next day. 72 hours later cells were fixed and analyzed by FACS. The percentage of cells with GFP signal from the empty vector sample was set to one (dotted line). The graph shows a representative image from three biological repeats (error bars=SEM). D) Phosphorylation of PALB2 supports RAD51 foci. Cells were transfected with siPALB2 overnight followed by forward transfection of siFBH1/siBLM for 6 hours then induced for expression of PALB2 (WT and TMA). 24 hours following induction cells were pulsed with EdU and exposed to HU as in Figure 3B. Cells in S-phase (EdU+) at the time of HU treatment were Click-IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ/Fiji (* p<0.0001, unpaired Student's t-test). Representative images of displaying RAD51 (green) and HA-PALB2 (red) localization in HU treated EdU positive (not shown) cells. Scale bar=5μm.
Figure 1(A) COUP-TFII mRNA levels in human kidney tissues of control (n=8) and CKD (n=53) subjects from GSE66494. ***p<0.001 by t-test. (B) COUP-TFII expression (green) is increased in human kidneys from patients with thrombotic microangiopathy (TMA) and diabetic nephropathy (DN) (n=2). Quantification by confocal micrographs in 400x hpf. ****p<0.0001 by one-way ANOVA; mean ± SD. The majority of these cells were localized in the interstitial region. More than 50% COUP-TFII (+) cells (green) are co-localized with expanded αSMA-positive (red) areas of fibrosis. Pearson correlation coefficient (PCC) = 0.56 ± 0.1 for patient#1 and 0.65 ± 0.13 for patient#2. (C) COUP-TFII expression (green) is increased in lungs from patients with IPF (n=2) by immunofluorescence. Quantification by confocal micrographs in 400x hpf. **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD. (D) Human kidney organoids were treated with IL-1β (10ng/ml) for 96 hours. Immunofluorescence reveals significantly increased COUP-TFII (green) and αSMA (red) expression in IL-1β treated organoids, compared to non-treated organoids. Quantification by confocal micrographs in 200x hpf. **p<0.01; ***p<0.001 by t test; mean ± SD. (E) COUP-TFII expression increased significantly in organoids treated with TGFβ1 (10ng/ml), cobalt chloride (100 μM), IL-1β (10ng/ml), but not PDGF (50ng/ml). Quantification by confocal micrographs in 400x hpf. *p<0.05; **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD. The higher magnification of the insert from IL-1β φησwed co-localization of COUP-TFII (green) and αSMA (red) staining.
a,b) Nocodazole flattens the differences in lysosomal mTOR localization and dampens mTORC1 signalling in response to changes in nutrient availability.figf2(a,b) Nocodazole flattens the differences in lysosomal mTOR localization and dampens mTORC1 signalling in response to changes in nutrient availability. Cells were treated as in Fig. 1a followed by incubation with dimethylsulphoxide (vehicle) or with nocodazole during the last 2 h before fixation/lysis. Samples were analysed by immunofluorescence (a) or immunoblotting (b). Quantification of phopho-S6K levels is shown in (b). (c-e) Changes in lysosomal positioning induced by kinesin- or small GTPase-family members (c,d) correlate with changes in mTORC1 activity (e). HeLa cells were transfected with overexpression constructs (OE) or with siRNA as shown, followed by immunofluorescence (c,d) or by immunoblotting (e) analyses. Values are means ± s.e.m. of three independent experiments carried out in triplicate. All comparisons are with the control within each treatment condition, *P0.05, ***P0.005 Student's t-test; n.s. not significant. Uncropped images of blots are shown in Supplementary Fig. S7.
x leads to increased interferon response and apoptosis.Figure 4A, Scatter plot of mean log2 mRNA fold-changes in the mutant cell lines versus control (n=3). Shown are also the Pearson correlation coefficient and respective P-value. The dashed line indicates equal change in the two mutant lines. B, Normalized enrichment score (ES) from gene set enrichment analysis (Subramanian et al, 2005) comparing gene expression changes in the mutant cell lines relative to the control cell line. For all depicted pathways, P < 0.05. P-values were calculated by comparing the empirical ES of a gene set to a null distribution of ESs derived from permuting the gene set, and then adjusted for multiple hypothesis testing. C, Mean (+/- s.d.) activity of STAT1/IRF1/IRF2/IRF3/IRF9 transcription factor motifs estimated by ISMARA (Balwierz et al, 2014) (n=3). Shown are the P-values (*** P < 0.001) determined by Dunn's multiple comparisons test post hoc and the non-parametric Kruskal-Wallis test (P < 0.0001). D, Mean (+/- s.e.m.) log2 mRNA expression fold-changes (inferred from RNA-Seq) of genes involved in the interferon alpha response and apoptosis in the two mutant cell lines relative to control (n=3). Multiple-testing-corrected P-values for Wald tests comparing fold-changes with respect to control are depicted above each bar (** P < 0.01, *** P < 0.001). E, Mean (+/- s.d.) expression fold-changes of the corresponding proteins (if detected in the proteomics data) in the two mutant cell lines (W1A, n=2; W6A, n=1) compared to Control (n=2). Multiple-testing-corrected P-values for unpaired two-tailed t-tests (proteomics) comparing fold-changes with respect to control are depicted above each bar (* P < 0.05, ** P < 0.01, *** P < 0.001). F, Representative result of the apoptosis assay using AnnexinV and propidium iodide (PI) staining in the control and the two mutant cell lines (left). The percentage of cells in each quadrant is depicted for each cell line. Quantification of the mean (+/- s.d.) percentage of apoptotic cells (Q1+Q2, AnnexinV+) across the different cell lines (n=4) (right). Shown is the P-value determined by unpaired two-tailed t-test. G, Impedance-based mean (+/- s.d.) cell index values at the indicated time points of growth after seeding equal numbers of control, W1A and W6A cells, upon treatment with DMSO or Ruxolitinib. For the DMSO treatment: Control (n=4), W1A (n=3) and W6A (n=3). For the Ruxolitinib treatment: Control (n=6), W1A (n=3) and W6A (n=4). From 6 hours after DMSO treatment, there is a statistically significant difference between control and the two mutants (P < 0.05, two tailed t-test), whereas no statistically significant difference is found after Ruxolitinib treatment.
Figure 4A) Cell cycle analysis (FACS) of C33 cell line after treatment with etoposide. The DNA contents of the cells were evaluated by propidium iodide staining, (PE-A; Phycoerythrin). The inset shows the % of the cells in each cell cycle phase. B) The cell cycle analysis of synchronised C33 cell line during the time. C) Evaluation of increasing concentrations of MDM2 on the cell cycle of C33 cell line after genotoxic stress induced with etoposide, (EV; empty vector). D) The same as in (C) but under normal cellular conditions. E) RB levels of the cells after 14 h of the release of nocodazole treatment. Etoposide was used to induce genotoxic stress. F) Evaluation of the G1 cell cycle phase after 14 h of the release of nocodazole treatment. Data information: Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05, **p <0.01).
Figure 1A Representative image of tumor-bearing mice and tumors after subcutaneous injection of Hepa1-6 hepatoma cells into WT and Tet2-/- (KO) mice. B Mean tumor volumes in WT and Tet2-/- mice (n=6). C Tumor weights 18 days after tumor cell injection into WT and KO mice (n=6). D Kaplan-Meier survival curves of tumor-bearing and tumor-free WT and KO mice (n=15). E Representative images of lungs with metastasis from WT and KO mice injected with Hepa1-6 cells via tail vein, as well as quantification of the number of lung metastasis (n=4). F Representative images of tumors from WT and KO mice subcutaneously injected with Py8119 breast cancer cells, as well as quantification of tumor weights (n=5).
Figure 5A. Transferrin (Tf, red) uptake in Syt11 KD (GFP-positive) and control neurons. Quantitative data are on the right. Scale bars, 20 μm. B. Tf uptake in Syt11 over-expressing and control neurons. Quantitative data are on the right. Scale bars, 20 μm. C. Normalized ∆Cm induced by 200-ms depolarization of a Syt11 KD neuron in the presence of 100 μM MDC was fitted to a double-exponential decay function (solid black and red, fitted curves). D. Fast and slow time-constants of endocytosis in Syt11 KD neurons recorded as in (C). E. KCl-evoked FM 1-43 uptake in DRG neurons after 1-h pre-incubation with 0.1% DMSO or 100 μM MDC in the bath solution.
a) RPE cells transiently co-transfected with GFP–myosin VI and cherry–LC3 and either left untreated (top row) or treated with 250 nM Torin1 for 2 h (bottom row) were processed for immunofluorescence microscopy.figf3(a) RPE cells transiently co-transfected with GFP-myosin VI and cherry-LC3 and either left untreated (top row) or treated with 250 nM Torin1 for 2 h (bottom row) were processed for immunofluorescence microscopy. The arrows highlight areas of co-localization and the insets show higher magnifications of the areas outlined in the main images. (b) RPE cells treated with 100 nM bafilomycin A1 were processed for immunofluorescence microscopy to evaluate endogenous myosin VI and endogenous LC3 co-localization. The arrows highlight areas of co-localization and the insets show higher magnifications of the areas outlined in the main images. (c) Image stills acquired from spinning-disc time-lapse video microscopy of RPE cells transiently co-transfected with cherry-LC3 and GFP-myosin VI. The outlined regions highlight areas of interest within the cell that are shown to the right (i-iii) as video image stills captured at 1-min intervals. The arrowheads highlight myosin-VI-positive vesicles (green) coming into contact and co-localizing with LC3-positive vesicles (red). (d) Confocal immunofluorescence microscopy of RPE cells stably expressing cherry-LC3 following transient transfection with GFP-myosin VI. Immunostaining for GFP (green) and cherry (red) was performed and actin was visualized with phalloidin (white). Nuclei were labelled with Hoechst (blue). The arrowheads indicate areas of actin-rich myosin-VI/LC3-positive autophagosomes. Scale bars, 20 μm (a,b,d); 10 μm (c, whole-cell image); 2 μm (c, cropped images).
Figure 2Structure-sequence analysis and context of the CSF-1R ΔA781_N783 and P824R variants. Sequences used in the alignment correspond to Uniprot sequences for CSF-1R as follows: Human P07333, Cynomolgus Monkey A0A2K5WG91, Mouse P09581, Bovine A7Z067, Cat P13369, Zebrafish Q9I8N6. Western blot for CSF-1R and Actin in HEK293 cells transfected with native and variant CSF-1R as indicated. Densitometry (top) and qPCR analysis of native and variant CSF-1R expression in HEK293 cells relative to native-transfected at 24 and 48 h (**** p < 0.0001, *** p < 0.0005, , ** p < 0.005 one-way ANOVA with Sidak's post-test for multiple comparison, n = 3 biological replicates, error bars indicate SEM). Western blot for phosphorylated and total ERK in HEK293 cells transfected with native and ΔA781_N783 (left) or P824R (right) CSF-1R and treated with CSF-1 (D, E) or IL-34 (F, G) for 10 minutes. Horizontal line indicates untreated cells, with increasing concentrations 10, 50 and 100 ng/ml. Corresponding densitometry displays the ratio of phospho-ERK to total ERK, normalised to the ratio of the untreated control for the native receptor (**** p < 0.0001, *** p < 0.0005, ** p < 0.01, * p < 0.05, one-way ANOVA with Sidak's post-test for multiple comparison, n = 3 biological replicates, , error bars indicate SEM). Western blot for CSF-1R in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with rapamycin. Horizontal line indicates untreated cells, with concentrations 0.1, 0.5, 1, 5, 10μM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (**** p < 0.0001, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM). Western blot for CSF-1R in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with 3-methyladenosine. Horizontal line indicates untreated cells, with increasing concentrations 0.1, 0.2, 0.5, 1mM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (**** p < 0.0001, * p = 0.0114, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM). Western blot for CSF-1R and Actin in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with MG132 or PLX3397. Horizontal line indicates untreated cells, 20μM PLX3397, with increasing concentrations 0.1, 0.5, 0.1, 5μM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (*** p = 0.0002, ** p = 0.002, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM).
Dipeptidyl-peptidase inhibition triggers a lytic cell death in human primary T cells (A and B) FACS-sorted T cell populations and Monocyte-derived Macrophages were treated with the indicated stimuli: Nigericin (Nig.Figure 1(A and B) FACS-sorted T cell populations and Monocyte-derived Macrophages were treated with the indicated stimuli: Nigericin (Nig. 4h), NeedleTox (4h), Val-boroPro (VbP 22h), ABT737/S63845 (A/S 22h). When indicated, cells were primed with LPS for 2 hours prior to stimulation. When indicated, MCC950 was added to the media 30 minutes prior to the addition of Nigericin. LDH-activity (A) and IL-1β and IL-18 concentration (B and C, respectively) in the supernatant were determined by LDH cytotoxicity assay and ELISA respectively. Individual data points ± SEM from three independent donors are shown. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; ns, not significant. P-values were corrected for multiple comparisons (Dunnett).
Atg36 drives mitophagy when directed to mitochondria.Figure 8(A)pex3Δ, pex3Δatg1Δ and pex3Δatg32Δ cells were grown for 6 h in galactose medium to express GAL1-Tom70-Pex3-mRFP and GAL1-Atg36-GFP. Cells were shifted to SD‐N medium and imaged after 12 h. Multiple fluorescent images were acquired in Z‐axis and flattened into a single image. Bright‐field image is a single plane. Bar, 5 μm. (B) OM45 was tagged with GFP in the genome of atg32Δpex3Δ and atg32Δpex3Δatg36Δ cells, which were transformed with an empty plasmid (−) or a plasmid encoding OM45-Pex3 (+). The cells were grown for up to 3 days in glycerol medium and samples were taken at 24 h intervals and processed for anti‐GFP western blotting. GFP* indicates the relative protease‐resistant degradation product and reflects vacuolar breakdown.
PLCβ1 and PTPRN2 drive metastatic migration and invasion.Figure 2(A) Matrigel invasion by 50,000 LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. Data normalized to control values. N = 6 inserts/group. (B) Migration assay by 100,000 LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. Data normalized to control values. N = 6 inserts/group. Right, representative images of the migration assay. Scale bar, 100 μm. (C) Quantification of area covered by cells 24 h after a scratch was made through confluent cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. N = 5 wells/group. Right, representative images of the scratch assay. (D,E) MDA-MB-231 cells transduced with PTPRN2, PTPRN2C945A, or control vector were subjected to the Matrigel invasion (D) and migration assays (E). N = 5 inserts/group. (F) Bioluminescence imaging quantification of lung colonization by 40,000 MDA-MB-231 cells overexpressing PTPRN2, PTPRN2C945A, or control vector. N = 5 mice/group. Right, H&amp;amp;E staining of representative lung sections. (G,H) MDA-MB-231 cells transduced with PLCβ1, PLCβ1H331Q, or control vector were subjected to the Matrigel invasion (G) and migration assays (H). N = 5 inserts/group. (I) Bioluminescence imaging quantification of lung colonization by 40,000 MDA-MB-231 cells overexpressing PLCβ1, PLCβ1H331Q, or control vector. For Cntrl and PLCβ1H331Q OE, N = 6 mice/group. For PLCβ1 OE, N = 5 mice. Right, H&amp;amp;E staining of representative lung sections.
Figure 7Schematic showing the anti-CD19 OptoCAR engineered to be light-responsive. Binding of the OptoCARCD19 expressed in T cells to CD19-expressing B cells drives signaling through an equivalent intracellular sequence used in the OptoCAR, where illumination with blue light causes the reversible disruption of receptor signaling. Stimulating primary CD4+ T cells expressing OptoCARCD19 over 24 hours in the dark drives expression of CD137 (4‑1BB), a robust activation marker. Continuous illumination completely abolishes this to CD137 levels equivalent to unstimulated T cells. Plot of geometric mean of CD137 expression when the length of an individual signaling pulse is varied. Single pulse ranged from 0 to 24 hours, with sample illumination initiated at the end of the pulse and CD137 expression measured for all samples 24 hours after initiation of signaling. Bars show mean ± SEM of biological replicates (n=4). The cRQ values from RT-qPCR of indicated cells. Total RNA was extracted and CD137 mRNA levels measured relative to that from HEK293T cells. Conjugated OptoCARCD19‑T cells were also assayed either before or after 24 hours stimulation in the dark state. Individual values from three biological replicates are shown, with mean depicted by bar plot. Series of experiments showing how CD137 expression is modulated by pulsatile trains of signaling in primary CD4+ T cells activated through OptoCARCD19. A combined signaling period of 6 hours was broken into pulses ranging from 15 minutes to 2 hours and the refractory period between these pulses varied (x-axis), shown in the schematic above each plot. Geometric mean of CD137 intensity is shown plotted as a function of inter-pulse interval, scaled to continuous 6-hour output. Bars show mean ± SEM (n=5). Representative dataset from (E), demonstrating how pulsatile stimulation of OptoCARCD19‑T cells drives more efficient activation. Dotted and gray histograms show CD137 expression in resting cells and 24-hour activation, respectively. The filled histogram shows either a continuous 6‑hour stimulation (left) or cumulative pulsed signals over 24 hours driving substantially more potent activation.
Figure 3Control or CEP41 siRNA-transfected HUVECs were immunostained with Ac-Tub- or GT335-specific antibodies in (A). The rectangles indicate the representative cells from each immunostaining experiment presented as magnified images in the right panels. Scale bars, 20 μm. Quantification of the ciliated cell numbers in the images in (A) from the results of three independent experiments with ≥ 200 cells per condition (mean ± SD) in (B). Wild-type and cep41-deficient Tg(kdrl:eGFP) zebrafish embryos were fixed for immunostaining with Ac-Tub- or GT335-specific antibodies at 28 hpf in (C). The rectangles indicate the arterial cilia (AC) and venous cilia (VC) in zebrafish endothelial cells (EC). Magnified representative images are displayed in the right panels. Scale bars, 40 μm. Quantification of the labeled cilia observed in equivalent fields of view are presented graphically in (D). Data are shown as median of three independent experiments (n ≥ 20 embryos per condition).