EMBO/sd-character-level-ner huggingface dataset:
Figure 2A. Representative images of immunofluorescence of Pan-hMENA (yellow) and α-SMA (red) in the primary PDAC tissue of patient #138 from whom high hMENA∆v6 CAFs were obtained. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 μm. The inset of the dashed area is provided on the right as a zoomed-in and cropped fluorescence image. Scale bar: 20 μm. αSMA‑positive CAFs are also positive for Pan-hMENA (arrow). B. Representative images of immunofluorescence of Pan-hMENA (yellow) and α-SMA (red) in NSCLC case #484 from whom high hMENA∆v6 CAFs were obtained. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 μm. The inset of the dashed area is provided on the right as a zoomed-in and cropped fluorescence image. Scale bar: 20 μm. As in A, α-SMA signal is evident in stromal cells which are also positive for Pan-hMENA (arrow). C. Boxplots showing the mean mRNA expression of ENAH gene (hMENA) for 22 patients in the 10 groups of cell types (Peng et al., 2019) (total number of cells: 38487, mean of cells for patient: 1603). Shown in each boxplot are the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line). Expressions from each cell-type group were compared to all other groups by using Mann-Whitney U-test (two-sided) and P-values were adjusted for multiple testing using the Benjamini-Hochberg method. Stromal cell-type groups with significantly up-regulated ENAH expression respect to other stromal groups are: Fibroblast, ***q=0.0002; Stellate, ***q= 1.5e-07. Non-stromal cell-type groups with significantly up-regulated ENAH expression with respect to other non-stromal groups are: Ductal 1, **q=0.0013. D. Boxplots showing the mRNA expression of ENAH gene (hMENA) in the 7 groups of cell types from (Lambrechts et al., 2018) (total n=52). Shown in each boxplot are the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line). Cell expression from each group were compared to all other stromal/not-stromal cells by using Mann-Whitney U-test (two-sided) and P-values were adjusted for multiple testing using the Benjamini-Hochberg method (Fibroblast group vs other stromal groups, **q=0.0022; Other comparisons, q>0.05
a) Kinetics of the appearance of P14 cells in spleens of C57BL/6 mice given adoptive transfer of 1 × 104 P14 cells, monitored after infection of recipients with LCMV Armstrong strain.figf1(a) Kinetics of the appearance of P14 cells in spleens of C57BL/6 mice given adoptive transfer of 1 × 104 P14 cells, monitored after infection of recipients with LCMV Armstrong strain. (b) Flow cytometry of cell size at various stages of P14 differentiation, assessed by forward scatter (FSC). (c) Immunoblot analysis of LC3b and p62 at various stages of P14 differentiation. Data are representative of two independent experiments with samples pooled from 5-20 mice for each time point.
A 20‐aa peptide binds to the extracellular domain of PRK5In vitro‐produced 35S‐labeled ectodomains of PRK5 (PRK540–281) or PRK4 (PRK440–279) were incubated with bacterially produced GST, GST‐GRI or GST‐GRI31–96 and purified.Figure 3A In vitro‐produced 35S‐labeled ectodomains of PRK5 (PRK540-281) or PRK4 (PRK440-279) were incubated with bacterially produced GST, GST‐GRI or GST‐GRI31-96 and purified. GST‐GRI31-96 but not GST directly bound to the ectodomain of PRK5. Binding of GST‐GRI to PRK5 ectodomain and binding of GST‐GRI31-96 and GST‐GRI to the ectodomain of PRK4 were strongly reduced. Upper part: autoradiograph, lower part: Coomassie‐stained 12% SDS-polyacrylamide gel. Asterisks in the Coomassie‐stained gel indicate GST, GST‐GRI and GST‐GRI31-96, respectively. Supplementary Fig S12 shows a Western blot of the GST‐tagged proteins. B Infiltration of 37 nM GST, GRIp31-96, GRIp65-84 or Y‐GRIp65-84 into Col‐0 or prk5‐2 leaves. Tyrosine‐labeled GRIp65-84 still induced cell death in Col‐0 but not in prk5‐2 plants. C 125I‐labeled Y‐GRIp65-84 (0.46 nM) bound specifically to Col‐0membrane fractions (light gray bars), the binding was significantly reduced in prk5 plants. Excess of non‐radioactive Y‐GRIp65-84 (10 μM) reduced binding to background levels (dark gray bars; all bars show the average of three samples, triangles show individual data points). D Immunoprecipitation of PRK5‐c‐myc, PRK4‐c‐myc or YFP expressed in protoplasts with rabbit polyclonal anti‐c‐myc antibody, immunoblotted with mouse monoclonal anti‐c‐myc antibody. E Binding of 125I‐Y‐GRIp65-84 (0.46 nM) to immunoprecipitates (using anti‐c‐myc antibodies) from prk5‐2 protoplasts transfected with PRK5‐c‐myc, PRK4‐c‐myc or YFP, respectively. Binding was competed out with 10 μM unlabeled Y‐GRIp65-84. Bars show the average of two samples, and triangles show individual data points. Western blot is shown in panel (D). F Analysis of 125I‐Y‐GRIp65-84 (0.46 nM) binding competed out with increasing amounts of unlabeled Y‐GRIp65-84 to Col‐0membrane extracts. Fifty percent inhibition (IC50) occurred at 25.2 nM. Red line shows binding average competition according to a sigmoid curve, dotted lines show 95% confidence intervals, and circles show data points. G Excess of GRIp65-84 (10 μM) but not of GRIp31-96 or other peptides (GRIp31-51, GRIp47-68, GRIp80-96) competed the binding of 0.46 nM 125I‐Y‐GRIp65-84 to membrane extracts from Col‐0 (all bars show the average of two samples, triangles show individual data points).
Mature B cells lacking Syk remain dependent on BAFF‐R for their survival in vivo and in vitroEx vivo survival assay performed on mature splenic B cells isolated from Tam‐treated mb1‐CreERT2 or mb1‐CreERT2;Sykfl/fl mice.Figure 6Ex vivo survival assay performed on mature splenicB cells isolated from Tam‐treated mb1‐CreERT2 or mb1‐CreERT2;Sykfl/flmice. The cells were cultured in the presence (dashed lines) or absence (solid lines) of 100 ng/ml human recombinant BAFF and harvested at the indicated time points. Viable cells were identified by flow cytometry using CD19 versus 7‐AAD staining. Shown are means ± SD from a triplicate experiment representative of at least three independent experiments. Histogram representation of the BAFF‐R surface staining on mature B cells from Tam‐treated mb1‐CreERT2;Sykfl/fl (solid line) or control mb1‐CreERT2mice (dashed line). Splenic transitional B cells derived from the BAFF‐R−/−mouse strain served as a negative control (dotted line). Quantitative analysis of absolute splenic B‐cell numbers from Tam‐treated mb1‐CreERT2 (filled circles) or mb1‐CreERT2;Sykfl/fl (open circles). The animals were treated either with the isotype control, non‐blocking anti‐BAFF‐R antibody clone 5A12 (open squares) or the anti‐BAFF‐R‐blocking antibody clone 9B9 (open diamonds). Mice were analyzed 2 weeks after antibody injection. Each circle represents an individual mouse. Absolute B‐cell numbers representing the different B lymphocyte subsets from different lymphoid organs are depicted. Each open circle represents an individual mouse from the mb1‐CreERT2;Sykfl/fl plus control Ab group while each open diamond a mouse from the mb1‐CreERT2;Sykfl/fl plus BAFF‐R‐blocking Ab group.
Fig.Figure 1A. qPCR data comparing the expression of the CamkIIδc transgene in the brain and heart of 3 month old CamkIIδc TG mice; n=4/group; *P <0.05, Unpaired t-test, two-tailed. Note that the transgene is not detected in the brain. B. Representative M-mode images from left ventricle from CamkIIδc TG and control mice. ESD: Left ventricle end systolic diameter, EDD: Left ventricle diastolic diameter. C. Ejection fraction, cardiac output and index are significantly decreased in CamkIIδc TG mice (n =8) when compared to control mice (n=5; *P <0.05, , **P<0.01, ***P<0.001, Unpaired t-test, two-tailed). Heart to body ratio, left ventricle to body ratio, and lung to body weight ratio are increased in CamkIIδc TG (n=8) compared to control (n=5; *P <0.05, **P<0.01, Unpaired t-test; two-tailed). D. Experimental scheme for RNA-seq analysis that was performed from hippocampal CA1 region of CamkIIδc TG mice (n=6) and control mice (n=5) at 3 months of age. Right panel shows principal component analysis (PCA) of the gene-expression data. The first principal component (PC1) can explain 42% of the variation between two groups. E. Volcano plot showing differentially expressed genes (FDR<0.05). Red color indicates up-regulation while green represents down-regulation of transcripts. F. Hypergeometric overlap analysis comparing genes deregulated in CamkIIδc TG mice to genes uniquely expressed in neurons, astrocytes or microglia. Numbers represent the P value after multiple adjustments with Benjamini-Hochberg (BH) method. Fisher's hypergeometric test; color represents fold enrichment. G. Dot plot showing Top GO biological processes after removing redundant GO terms using Rivago. H. qPCR quantification of selected genes reflecting ER-stress or protein methylation-related processes in CamkIIδc TG mice (n=4) and control mice (n=5). *p<0.05, ns = non-significant, Unpaired t-test; two-tailed. Data is normalized to Hprt1 expression. Hypergeometric overlap analysis comparing genes deregulated in CamkIIδc TG mice to genes deregulated under (I) hypoxia conditions Benjamini-Hochberg (BH) adjusted P values after are denoted as numbers and fold enrichment is represented as color heatmap. Fisher's hypergeometric test. Data information: Bars and error bars indicate average ± standard error mean. Hypergeometric overlap analysis comparing genes deregulated in CamkIIδc TG mice to genes deregulated under (J) in response to tunicamycin-induced ER-stress Benjamini-Hochberg (BH) adjusted P values after are denoted as numbers and fold enrichment is represented as color heatmap. Fisher's hypergeometric test. Data information: Bars and error bars indicate average ± standard error mean. Hypergeometric overlap analysis comparing genes deregulated in CamkIIδc TG mice to genes deregulated under (K) in hippocampal tissue from animal models of memory impairment and neurodegeneration. Benjamini-Hochberg (BH) adjusted P values after are denoted as numbers and fold enrichment is represented as color heatmap. Fisher's hypergeometric test. Data information: Bars and error bars indicate average ± standard error mean.
Figure 1(A) Frequency of CHD1 gene mutation (green) deep deletion (blue) or amplification (red) in prostate cancer patients. (B) CHD1 is recruited to an I-SceI-induced DSB site and is co-localized with γH2AX. Immunofluorescence studies using U2OS19 ptight13 GFP-LacR cells containing a stably integrated I-SceI cleavage site flanked by 256 copies of lac operator (lacO) on one side and 96 copies of the tetracycline response element on the other side (tetO). The localization of the GFP-lac repressor protein (GFP-LacR) at the lac-operator DNA sequences in the nucleus before (− I-SceI) and 16 h after I-SceI-induced (+ I-SceI) DSB. After 16 h of doxy treatment, CHD1 and γH2AX co-localized at I-SceI cleavage site, along with DNA-bound GFP-LacR but not in uninduced cells (−I-SceI). Scale bar 10 µm. (C) Quantification of co-localization of CHD1 with the lac array, mean values of three independent experiments counting at least 100 cells are represented in the graph as ± SD. (D) PC3 cells were treated with NCS (100 ng/ml) for 2 h and with EdU for 45 min. Cells were stained with EdU first and then proximity ligation assay (PLA) was performed using γH2AX and CHD1 antibodies. Scale bar 10 µm. (E) Quantification of PLA signal from (D) in EdU-positive and -negative cells using ImageJ. For quantification more than 100 cells were analyzed for each condition and represented as mean value (n=3, ± SD). (F) CHD1 is recruited to the chromatin upon DNA double strand break induction. PC3 cells with stable control (shCont) or CHD1 shRNA (shCHD1) expression were treated with NCS for the indicated time points and chromatin fractions were immunoblotted with CHD1 and γH2AX antibodies. H2B was used as a loading control. See also Figure EV1A-EV1F.
Loss of GRK2 impairs canonical Wnt signaling.Figure 6(A) Control and GRK2-/- fibroblasts were transfected with the TOPflash Firefly (F) luciferase vector together with a control Renilla (R) luciferase vector, and the effect of Wnt3A on TOPflash transcriptional activation was determined by dual-luciferase assay. Note the impaired response to Wnt3A in GRK2-/- cells. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated. (B Low phosphorylation (p) of Wnt3A co-receptor LRP6 as determined by western blot with pS1490- and pT1572-LRP6 specific antibodies, reduced LRP6 and DVL2 expression, and increased steady state phosphorylation of DVL2. A representative experiment of four is shown. #, nonspecific band. C) Low phosphorylation (p) of Wnt3A co-receptor LRP6 as determined by western blot with pS1490- and pT1572-LRP6 specific antibodies, reduced LRP6 and DVL2 expression, and increased steady state phosphorylation of DVL2. (C) Densitometry of western blot results demonstrating decreased levels of LRP6 in GRK2-/- cells, lower response of GRK2-/- cells to Wnt3A by LRP6 phosphorylation at S1490, and tendency towards the similar defect at T1572. Densitometry also showed less of total DVL2 levels, and increased steady state pDVL2 in GRK2-/- cells. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated. (D) Loss of interaction between Frizzled 4 (FZD4) and ß-Arrestin 2 (ARRB2) in GRK2-/- fibroblasts. Proximity ligation assay (PLA) between expressed FZD4-GFP and ARRB2-Flag showed significantly reduced proximity events between the two proteins in GRK2-/- cells. White dashed lines outline the transfected cells. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cells are indicated. Scale bar, 10 µm.
Figure 3(A) Immunofluorescence (IF) analysis with the indicated antibodies in sections from murine small intestine of 2-month-old WT mice. (B) Double IF analysis with the indicated antibodies in sections from murine small intestine of 2-month-old Lgr5-GFP transgenic mice. (C) Western Blot analysis of chromatin extracts from isolated intestinal villus and crypt cells. (D) Western Blot analysis of chromatin extracts from isolated intestinal villus and crypt cells in several 2-month-old WT mice (each # represents a mouse). (E) Western Blot analysis of soluble (Sol) and chromatin (Chr) extracts from isolated intestinal villus and crypt cells in 2-month-old mCherry-H4 transgenic mice. (F) Western Blot analysis of chromatin fraction from mCherry-H4 transfected HCT-116 cells. (G) Pulldown assay using GST-SUMO IκBα protein and chromatin lysates from 2-month-old WT mice villi.
Figure 9A Confocal images of liver cryosections from Li-CluhWT and Li-CluhKO mice injected with rapamycin or mock solution and stained with anti-TOM20 antibody. Scale bar, 25 µm. B Quantification of morphological parameters of experiments shown in (A). 4 images were quantified per animal and averaged. Nuclear staining was excluded for the analysis (n=3 mice per genotype).Data information: data are presented as histograms showing the mean ± SEM. **, P≤0.01; *, P≤0.05 (Student's t-test). C Confocal images of liver cryosections from Li-CluhWT and Li-CluhKO mice injected with rapamycin or mock solution and stained with anti-LAMP1 antibody. Scale bar, 25 µm. D Quantification of morphological parameters of experiments shown in (C). 4 images were quantified per animal and averaged. Nuclear staining was excluded for the analysis (n=3 mice per genotype). Data information: , data are presented as histograms showing the mean ± SEM. **, P≤0.01; *, P≤0.05 (Student's t-test). E Confocal images of COS-7 cells downregulated for the indicated genes and stained with anti-TOM20 antibody. Scale bar, 10 µm. F Representative western blot of COS-7 cells downregulated for the indicated genes of experiments and probed with indicated antibodies. Asterisks indicate unspecific signal or signal of previous incubation. G Quantification of morphological parameters of experiments (n>100 cells from 3 independent experiments). Data information: In (G), data presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. (G) ***, P≤0.001; **, P≤0.01 *, P≤0.05 (One-way ANOVA, Tukey's multiple comparisons test).
a) rho::GFP-tau.f9(a) rho::GFP-tau fishes were incubated from 3 to 9 d.p.f. in either dimethyl sulphoxide (DMSO) or rapamycin (i) or from 3 to 7 d.p.f in EM alone or 10 mM ammonium chloride or 100 nM Wortmannin (ii). Images through the central retina at 9 d.p.f. (top panel; (i)) reveal rod degeneration (arrow) throughout the retina in control (DMSO-treated) larvae, whereas rod photoreceptors are present throughout the retina, particularly in the central region following treatment with rapamycin (arrow). Images taken through the central retina at 7 d.p.f. (bottom panel; (ii)) show normal photoreceptors in the marginal zones (arrows) and only limited numbers in the central region. NH4Cl exacerbates degeneration-photoreceptors are absent from the central retina and reduced/absent from marginal zones (arrows). Sections were stained with anti-rhodopsin (1D1) antibody. GFP labels whole rod photoreceptors, whereas rhodopsin is present in the rod outer segment. GFP co-localizes with the red rhodopsin label in all experimental conditions. Scale bars, 50 μm. Quantification of rodphotoreceptor degeneration (n=10 larvae per group; ***P0.001; ###P0.001, two-tailed unpaired t-test). Error bars are mean ±s.d. (b) Full-length CALM electroporation into rho::GFP larvae did not cause degeneration., while full-length CALM electroporation into rho::GFP-tau larvae exacerbated photoreceptor degeneration. Central retina sections in the region of the optic nerve head are presented. Scale bars, 50 μm. Data represent the ratio (in %) of the intensity of the GFP signal between CALM-HA electroporated eye versus control eye for five individuals per transgenic line. ***P0.05; two-tailed t-test. Error bars are mean ±s.d. (c) NH4Cl treatment of rho::GFP-tau immediately after unilateral CALM electroporation caused photoreceptor degenerationon the control (non-electroporated) side but did not alter the degeneration caused by CALM electroporation. Rapamycin treatment of Rapamycin::GFP-tau immediately after unilateral CALM electroporation rescued photoreceptors on the control (non-electroporated) side but not on the CALM-electroporated side. To demonstrate that loss of GFP corresponds to loss of photoreceptors, sections were stained with anti-rhodopsin (1D1) antibody. Wilcoxon signed rank test was used to compare the left eye versus the right eye of the same fish; Mann-Whitney test was used to compare drug treatment in the right eye of different fishes. *P0.05. Scale bars, 50 μm. Error bars are mean ±s.e.m.
Figure 2(A) The schematic diagram showing two potential Ets1 binding sites in the Islr promoter. (B) qRT-PCR analysis for Ets1 in colonic epithelium and lamina propria. n = 3. (C) Double immunofluorescence for ETS1 and β-catenin in intestinal biopsies of healthy controls and inflamed mucosa from UC patients. Scale bar: 50 μm. (D) qRT-PCR for Ets1 in colons with or without 5-day DSS treatment. n = 4. (E) Double immunofluorescence for Ets1 and β-catenin in normal mouse colons without DSS treatment, mouse colons with 5-day DSS treatments, and AOM-DSS mouse colon tumors. Scale bar: 50 μm. (F) Pearson correlation analysis on ETS1 and ISLR in colorectal adenocarcinoma TCGA RNA-seq (P < 0.001; R = 0.68). (G) Western blotting for ISLR and HA in lysates of UC-MSCs transfected with pCMV5-HA or pCMV5-ETS1. α-Tubulin was used as a loading control. (H) Luciferase activity in lysates of UC-MSCs transfected with luciferase reporter plasmids of pGL3-basic empty vector (basic), wild type ISLR promoter or mutant promoter with mutation of ETS1 binding sites under normal and ETS1 overexpression conditions. n = 3. (I) Chromatin immunoprecipitation assay was carried out on UC-MSCs cells using antibodies against ETS1 and Histone H3. The antibody against Histone H3 was used as a positive control. IgG was used as a negative control. The enrichment of ETS1 binding to ISLR promoter was quantified using qPCR. n = 3 technical replicates. (J) Chromatin immunoprecipitation assay was carried out on primary intestinal stromal cells isolated from WT mice without DSS treatment or WT mice after 5-day DSS treatment using antibodies against ETS1 and Histone H3. The antibody against Histone H3 was used as a positive control. IgG was used as a negative control. The enrichment of Ets1 binding to Islr promoter was quantified using qPCR. n = 3 technical replicates.
Red1 promotes the destabilization of DSR‐containing mRNA.Figure 5(A) Schematic representation of the ura4+‐DSR construct. The DSR region derived from mei4+ was fused to the ura4+ gene and this ura4+‐DSR mRNA was driven by the nmt81 promoter, which was repressed in the presence of thiamine. This construct was integrated into the lys1+ locus. (B) red1Δ led to the 'ura4+‐on' state in cells carrying the ura4+‐DSR construct. Serial dilutions of wild‐type (WT), red1Δ, and mmi1‐619 (mmi1) cells containing the ura4+‐DSR construct were spotted onto a complete or uracil‐lacking plate and then incubated for 3-4 days at 30°C. (C) The ura4+‐DSR transcript accumulates in red1Δ and mmi1‐619 cells. Wild‐type, red1Δ, and mmi1‐619 cells carrying both the ura4+‐DSR construct and a ura4+ minigene (ura4DS/E) were grown in the absence of thiamine and then subjected to RT-PCR analysis using a ura4+ primer set that amplifies both ura4+‐DSR and ura4DS/E. The ura4DS/E was used as the internal control. (D) red1Δ does not increase RNA polymerase II (Pol II) occupancy or the levels of histone H3 Lys14 acetylation (H3K14ac). Wild‐type, red1Δ, and mmi1‐619 cells expressing both ura4+‐DSR and ura4DS/E were subjected to chromatin immunoprecipitation using anti‐Pol II and anti‐H3K14ac antibodies. The precipitated DNAs were analysed by PCR using the same primer described in (C). (E) The ura4+‐DSR became more stable in red1Δ compared with wild‐type cells. Thiamine was added to the culture of wild‐type or red1Δ cells grown without thiamine, and then the levels of ura4+‐DSR and ura4DS/E transcripts were examined by RT-PCR.
Figure 4(A) H1299 cells were co-transfected with the indicated expression vectors (p53, 7.5 ng; HA-Ub, 0.5 ug; His-Xpress-MDM2, 2 ug; RPL11-FLAG, 1 ug; HA-GRWD1-FLAG, 1.5 ug; GFP, 0.04 ug) for 48 h and analyzed by immunoblotting with the indicated antibodies. GFP serves as a control protein to show equal transfection efficiencies. (B) Lysates were prepared from 293T cells co-transfected with His-Xpress-MDM2 (1.2 ug), FLAG-RPL11 (1.8 ug), and HA-GRWD1 (1 ug) as indicated for 42 h and then immunoprecipitated with anti-FLAG antibody. Immunoprecipitates (IPs) and 1.5% of inputs were immunoblotted with the indicated antibodies. (C) In vivo ubiquitination assay to detect MDM2 autoubiquitination in H1299 cells. Lysates were prepared from H1299 cells transfected with the indicated expression vectors (His-Xpress-MDM2, 2 ug; HA-Ub, 0.5 ug; RPL11-FLAG, 1 ug; HA-GRWD1-FLAG, 1.5 ug) for 48 h, treated with proteasome inhibitors for 6 h before harvest, and then immunoprecipitated with anti-MDM2 antibody. Immunoprecipitates (IPs) and inputs were immunoblotted with the indicated antibodies. (D) In vitro ubiquitination of p53 by immunopurified MDM2. His-Xpress-MDM2 was immunopurified from transfected 293T cells with anti-Omni probe antibody. Recombinant p53 was incubated with E1, E2 (UbcH5a), His-ubiquitin, ATP, GST-RPL11, GRWD1-His, and immunopurified His-Xpress-MDM2 or control immunoprecipitates at 30℃ for 120 min as indicated. The samples were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies.