EMBO/sd-character-level-ner huggingface dataset:
Figure 2Schematic diagram of the 3" end of the Prdm1ihCd2 allele (Minnich et al., 2016). The frt-flanked IRES-hCd2 (ihCD2) reporter gene, which was linked to a 258-bp long terminal repeat (LTR) sequence of the Moloney murine leukemia virus (MoMLV, green), was inserted between the stop codon and 3" UTR of the Prdm1 gene. C-terminal sequences of exon 8 contained in-frame tag sequences encoding the Flag and V5 epitopes, two TEV protease cleavage sites and a biotin acceptor sequence. The inserted MoMLV enhancer sequence is shown below together with its regulatory elements (Speck et al., 1990). GRE, glucocorticoid response element; LV, leukemia virus factor-binding site; NF1, nuclear factor 1; polyadenylatio Expression of Prdm1 and Atg5 mRNA in ex vivo sorted pro-B and pre-B cells from the bone marrow of Prdm1ihCd2/+ or wild-type (WT) mice. mRNA expression is shown as mean expression value (TPM) with SEM based on two independent RNA-seq experiments for each cell type and genotype RT-qPCR analysis of nascent Prdm1 and Atg5 transcripts in ex vivo sorted pre-B cells and in in vitro differentiated plasmablasts (PB) of the indicated genotypes. Plasmablasts were generated by stimulation of splenic FO B cells for 4 days with LPS. The data were normalized to those of the ubiquitously expressed control gene Tbp and are presented relative to those of the wild-type pre-B cells (set as 1). Nascent transcripts were PCR-amplified with primers located in the indicated introns (Table EV3). Statistical data are shown as mean value with SEM and were analyzed by the Student"s t-test; *P < 0.05, **P < 0.01. Each dot corresponds to one Flow cytometric analysis of hCD2 cell surface expression and intracellular Blimp1 expression of the indicated cell types. Bone marrow of Prdm1ihCd2/+ (red) or wild-type (WT, gray) mice was used to analyz ALPs, BLPs, pro-B, pre-B, immature B cells whereas the spleen was used for flow cytometric analysis o FO B, MZ B and plasma cell of both genotypes The histograms show hCD2 (top row) and Blimp1 (bottom row) expression for the different cell types Wild-type FO B cells (dashed line) were used as negative control for the Blimp1 staining in plasma cells. The difference in mean fluorescence intensity (ΔMFI) between the two genotypes is shown for each cell type Flow cytometric analysis of hCD2 cell surface expression and intracellular Blimp1 expression of the indicated cell types. Bone marrow of Prdm1ihCd2/+ (red) or wild-type (WT, gray) mice was used to analyz NK cells and granulocytes, whereas the spleen was used for flow cytometric analysis o naïve CD4 T and naïve CD8 T cells of both genotypes The histograms show hCD2 (top row) and Blimp1 (bottom row) expression for the different cell types The difference in mean fluorescence intensity (ΔMFI) between the two genotypes is shown for each cell type Immunoblot analysis of Blimp1 and the TATA-binding protein (TBP) in nuclear extracts prepared from short-term cultured wild-type or Prdm1ihCd2/+ pro-B cells. Marker proteins of the indicated size (in kilodaltons) are shown to the right
Figure 1Schematic representation of the experimental design Representative images of HeLa (B) or HCT-8 (C) cells infected with Shigella WT pre-treated or not with arsenite, analyzed at the indicated times post-infection Cfu quantification of intracellular bacteria in HeLa and HCT-8 cells pre-treated or not with arsenite and infected with Shigella Representative images (E) and cfu quantification (F) of intracellular bacteria in HeLa cells infected with Shigella WT after pre-treatment with TNF-α, H2O2, anisomycin, hypoxia and corresponding controls, analyzed at 0.5 hpi Representative images (G) and cfu quantification (H) of intracellular Shigella in HeLa cells pre-treated with arsenite, anisomycin, stressors plus NAC, and corresponding controls
Figure 6A) EGFR activation in the ADAM17 variant expressing DLD-1 Adam17-/- cells upon H2O2 treatment, determined by Western blot and quantified below as the ratio of EGFR autophosphorylated at Tyr1068 to total EGFR. Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05, **p<0.01. Mean and standard deviation indicated from three independent experiments. (B, C) B) Cell proliferation and C) Matrigel invasion of the ADAM17 variant expressing DLD-1 Adam17-/- cells. Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05, **p<0.01. Mean and standard deviation indicated from three independent experiments. D) Protein expression of the ADAM17 variants in the mouse breast cancer cell line 4T1 Adam17-/-, determined by Western blot. GAPDH was used as an internal loading control. E) Average tumor volume ± SEM of 4T1 Adam17-/- cells expressing the ADAM17 variants injected into the mammary fat pad of Balb/c mice (n=10 for each group). Indicated significances are between the I762A and LEE tumors. Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05. Mean and standard error of the mean indicated. F) Survival curve of the tumor bearing mice injected with 4T1 Adam17-/- cells expressing the ADAM17 variants (n=10 for each group). The indicated significances are between the LEE and wt tumor bearing mice, and the LEE and I762A tumor bearing mice. Log-rank test was applied to test for significant differences. *p<0.05.
Figure 6A. Representative maximum-intensity projected coherent-hybrid STED (CH-STED) images of mitotic spindles in U2OS cells treated with control and NDC80 siRNAs and stained for DNA (DAPI), KTs (CENP-C, magenta), α-tubulin (red) and CENP-E (green). Scale bar, 10 µm. B. Model illustrating potential mechanisms for CENP-E mediated MT-flux forces either through its KT- or antiparallel MT- based localization and sliding. CENP-E localized at KTs laterally interacting with MTs (white arrowheads in 1, 2), and CENP-E localized at interpolar MTs (white arrows in 3, 4), in control and NDC80-depleted mitotic cells. Scale bars, 2 µm. C. CH-STED analyses of HeLa cells stably expressing CENP-E-GFP illustrating bipolar spindles in prometaphase, metaphase and just after sister-chromatid separation at anaphase onset. Approximate x-y resolution is 70 nm. The expected/described localization of CENP-E-GFP (green) at KTs is shown. In addition, CENP-E-GFP was found associated with interpolar MTs (ipMTs), including regions of overlapping anti-parallel MTs, and k-fibers. Chromosomes (confocal mode only) were revealed in the larger panels with DAPI (cyan) and MTs (magenta) were detected with an anti-α-tubulin antibody. Arrows indicate examples of clear MT bundles, including regions of overlapping anti-parallel MTs. Scale bar in all panels is 5 μm. D. Representative spinning disk confocal live-cell time series of HeLa cells stably expressing CENP-E-GFP. White arrowheads highlight CENP-E-GFP at KTs, white arrows highlight CENP-E-GFP on interpolar MTs. Scale bar, 10 µm. Time, hour:min.
Human stroma is lost after engraftment and replaced by murine cells.Figure 3Stromal elements of the primary tumors were labeled with anti-humanCD34 but not anti-mouseCD34; patient-derived xenografts are labeled with anti-mouseCD34 only and no anti-human marker. Representative sections (at 40× and 400× magnification) of the primary tumors (PT) and the orthoxenograft tumors at passages 1 (OT P1) and 4 (OT P4) were labeled with anti-humanCD34 (H) and anti-mouseCD34 (M). Sanger sequencing of the germline NF1 mutation c.350T>A present in the patient that developed two different tumors (MPNST-NF1-001 and MPNST-NF1-002). Sequencing results from normal tissue, primary tumor (PT), and orthotopic xenograft tumors (OT) are shown. The sequence of the NF1 region revealed WT NF1 alleles in primary tumor samples, and not in the corresponding derived orthoxenografts, indicating probable loss of the human stromal cells. SNP array of primary tumor (PT) and orthotopic xenograft passages 1 (OT P1) and 4 (OT P4) for MPNST-NF1-001 and MPNST-NF1-002 tumors. Results correspond to chromosome 17. Images show the B-allele frequency (BAF) for the different samples as scatter plots and the copy number callings below them represented by thick horizontal lines: 2n regions are shown in gray, gained regions in orange, lost regions in green, and LOH regions are represented in blue. The vertical red line indicates the location of the NF1 locus. In addition, the percentage of tumor versus stromal cells for each sample is represented in a pie chart (blue, stromal cells; red, tumor cells).
Figure 1Time-lapse imaging of an oocyte expressing CENP-C-EGFP (centromeres, green) and H2B-mCherry (chromatin, red). Upper row shows maximum intensity z projection images from representative timepoints of four stages from anaphase I onset to CSF-dependent MII arrest. The 3D positions of centromeres are shown as black dots in a side view (along the spindle equator, middle row) and top view (perpendicular to the equator plane, bottom row). Tracks of individual centromeres are color-coded according to the speed, as indicated by the colour bar. Time is shown after anaphase I onset (min:sec). Scale bars, 5 μm.
Figure 4(A-C) Representative images for detection of ATM pS9181, γH2AX, and 53BP1 at telomeres in wild-type (WT) and SMCHD1 knockout HeLa cells transfected with shTRF2 plasmid and empty vector (EV) control. Immunofluoresence (IF) for ATM pS1981 (gray), γH2AX (green) and 53BP1 (yellow) was combined with telomeric (CCCTAA)3-FISH (red) and DAPI staining total DNA. Scale bars: 5µm (D) Quantification of the number of cells containing >5 Telomere dysfunction Induced Foci (TIFs) detected Data represent the mean of 4 independent experiments ± SD (>200 cells/condition/experiment) for ATM pS1981 and 3 independent experiments ± SD (>200 cells/condition/experiment) for γH2AX and 53BP1. (***) P < 0.001; unpaired two-tailed Student's t-test. (E) Quantification of the number of 53BP1 TIFs per cell detected from three independent experiments. Data represent individual values per cell with the bar representing mean ± SD. (***) P < 0.001; unpaired two-tailed Student's t-test.
a) Diagram of Fbxo7 isoforms 1 and 2, showing the location of functional domains and the Parkinson's disease–associated mutations.figf1(a) Diagram of Fbxo7 isoforms 1 and 2, showing the location of functional domains and the Parkinson's disease-associated mutations. (b) Coimmuno-precipitation (IP) of Fbxo7-HA and Flag-Parkin in whole-cell lysates from U2OS cells over-expressing both proteins. IB, immunoblot. Molecular mass markers are in kilodaltons. (c) Coimmunoprecipitation of endogenous Fbxo7 with Flag-Parkin in HEK293T cells transfected with Flag-Parkin or a control protein (EGFP). (d) Flag immunoprecipitation was repeated in U2OS cells transfected with Flag-Parkin and either full-length (1-522) T7-Fbxo7 or an N-terminal truncation lacking the Ubl domain (89-522). (e) As in d, using lysates from U2OS cells expressing Flag-Parkin and T7-Fbxo7, either full length (1-522) or a C-terminal deletion of the proline-rich region (1-398). * indicates IgG heavy chain. (f) In vitro-translated Flag-Parkin was incubated with bacterially expressed GST or GST fused to the Fbxo7 Ubl domain (1-88) immobilized on glutathione beads. Bead-bound proteins and inputs were analyzed by immunoblotting with anti-Parkin antibodies. (g) The disease-causing mutation T22M interferes with Fbxo7's interaction with Parkin. Coimmunoprecipitation was performed as in b using lysates from U2OS cells expressing Flag-Parkin and WT or T22M Fbxo7-HA. (h) Coimmunoprecipitation of Flag-Parkin and Fbxo7 in the mitochondrial and cytosolic fractions of HEK293T cells overexpressing both proteins. CxVα and GAPDH are markers for mitochondria and cytoplasm, respectively. All western blots are representative of experiments performed at least three times. Full-length blots are presented in Supplementary Figure 9.
DNA methylation analysis of iPSC‐derived DAn from monogenic LRRK2‐associated PD (L2PD) and sporadic PD (sPD) patients across the cell types involved in the cell reprogramming and differentiation processes.Figure 2Principal component analysis (PCA) of methylation data from 28,363 CpG sites with variable methylation values (SD > 0.1) in iPSC‐derived DAn cell lines (n = 14). This analysis shows different DNA methylation profiles between PD patients and controls, and no differences between L2PD and sPD. PC, principal component.Bar diagram showing the number of differentially methylated CpGs (DMCpGs) among iPSC‐derived DAn lines (n = 14) as detected in multiple comparisons. This analysis indicates that L2PD and sPD share similar DNA methylation changes with respect to controls. Hypermeth, hypermethylation; hypometh, hypomethylation.Relative distribution of DMCpGs in PD iPSC‐derived DAn lines (n = 10) across inter‐ and intragenic regions (left), across different gene‐related regions (middle), and within CpG islands (CGI), CGI shores, CGI shelves, or outside CGI (right). Bkg, background (methylation platform). DNA methylation analysis across the cell types involved in the cell reprogramming and differentiation processes and also iPSC‐derived neural cultures not‐enriched‐in‐DAn. Scheme of the cell reprogramming and differentiation protocols (upper part of the panel). Heatmaps and density color code of the 2,087 DMCpGs identified in iPSC‐derived DAn from PD patients with respect to controls (n = 14), as well as their methylation status in parental fibroblasts (n = 9), undifferentiated iPSCs (n = 9), and iPSC‐derived neurons (not‐DAn) (n = 9) (lower part of the panel) (Wilcoxon rank test for independent samples with delta‐beta above ¦0.25¦ and FDR‐adjusted P < 0.05).
Optic nerve myelination is affected by reduced Nr2f1 dosage.Figure 4(A-C) NF1A (red, astrocytes) and Sox10 (green, oligodendrocytes) IF on cross-sections of WT and KO P8 ONs showing altered ratio of astrocytes and oligodendrocytes in mutants, particularly in KOs, as quantified in (C). data are represented as means ± SEM. N=4-5 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001 Scale bars: 50µm (D-J) MBP (yellow, fully differentiated oligodendrocytes) IF on cross-sections of WT, HET and KO P7 optic nerves (ONs) at the chiasm (D-F) and between the chiasm and the eye (G-I). Note the dramatic decrease of MBP+ oligodendrocytes at the chiasm in HET and KO, quantified in (J). The ratio of MBP+ cells is calculated over the total DAPI+ cell number per chiasmal/ON section. data are represented as means ± SEM. N=4-5 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm (K-N) EM thin sections of P8 WT, HET and KO ONs depicting myelin as a dense, dark staining around axonal fibers illustrate a significantly lower number of myelinated fibers in HET and KO, as quantified in (N). Higher magnification images (insets) showing different degrees of myelination in HET and KO ONs. data are represented as means ± SEM. N=3 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm (4µm) TEM images of P28 WT and HET confirming persistent hypomyelination in mutants at P28 Scale bars: (4µm) P28 WT and HET confirming persistent hypomyelination in mutants at P28, as quantified in (Q). data are represented as means ± SEM. N=3 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm (R,S) High magnification EM images of P28 WT and HET ONs showing the difference in myelin compaction between WT (arrowheads in R) and HET (arrowheads in S). Scale bars: (1µm).
Figure 2(A) Overall structure of complex showing Ubp8 (green) with ubiquitin (yellow) bound to the USP domain. (B) Hydrogen bonding contacts between the C-terminal carboxylate of ubiquitin and Ubp8. (C) In grey spheres, van der Waals radii of C146 (teal) and A146 (green) in steric proximity of ubiquitin's C-terminal carboxylate (yellow). DUBm-Ubp8WT structure is shown in teal (PDB ID 3MHH) and DUBm-Ubp8C146A is shown in green.
AV types involved in FP+.Figure 8AV types involved in FP+. The same FP+ data as used in Fig. 6 were classified according to the AV types.
a) Induction of GFP+ dots in differentiated THP-1 cells stably expressing GFP-LC3, left untransfected or transfected for 6 h with poly(dA:dT) (1.figf1(a) Induction of GFP+ dots in differentiated THP-1 cells stably expressing GFP-LC3, left untransfected or transfected for 6 h with poly(dA:dT) (1.5 μg/ml), then exposed to control antibody (Control Ab), antibody to neutralize IL-1β (Anti-IL-1β), dimethyl sulfoxide (DMSO) or 3-MA (5 mM); results are presented as quantification of GFP-LC3+ dots (top) and images of individual cells (middle). Scale bars, 10 μm. (b) Expression of AIM2 and beclin-1 in cells transfected with control siRNA or siRNA targeting AIM2 or beclin-1; numbers below lanes indicate band intensity (ratio of cells transfected with targeting siRNA to those transfected with control siRNA). (c) Induction of GFP+ dots in differentiated THP-1 cells stably expressing GFP-LC3 and treated with various siRNAs (below graph), quantified 6 h after transfection with poly(dA:dT) (+) or no transfection (-). (d) Immunoblot analysis of the expression of LC3-I and LC3-II (above) in wild-type (WT) and Aim2−/− BMDMs left untransfected or transfected for 6 h with poly(dA:dT); numbers below lanes indicate division of the ratio of induced LC3-I to induced LC3-II by the ratio of basal LC3-I to basal LC3-II (band intensities). Below, immunoblot analysis of IL-1β in supernatants after overnight culture. (e) Immunoblot analysis of LC3-I and LC3-II in or wild-type, ASC-deficient (Pycard-/-; called 'Asc-/-' here) or caspase-1-deficient (Casp1-/-) BMDMs primed with LPS (20 ng/ml) and then left untransfected or transfected with poly(dA:dT) or left untreated or treated with nigericin for 4 h; numbers below lanes as in d. Actin serves as a loading control throughout. NS, not significant; *P 0.05 and **P 0.01 (unpaired t-test). Data are from three individual experiments
Figure 5A. 293T cells were transfected with poly(I:C) and cell lysates were prepared at the indicated times for co-immunoprecipitation analysis of endogenous HDAC6 and RIG-I. Actin was used as the loading control. B. BMDMs were isolated from HDAC6+/+ and HDAC6-/- mice and were transfected with 5ˊppp-dsRNA. Whole cell lysates were prepared after 8 h of transfection and co-immunoprecipitation analysis of RIG-I and HDAC6 was performed. C. HeLa cells were transfected with 5ˊppp-dsRNA. Cells were stained with anti-RIG-I (red) and anti-HDAC6 H300 (green) antibodies after 8 h of transfection. Nuclei were stained with DAPI (blue) (bar, 20µm). D. Fluorescence microscopy, green fluorescence absorbance and virus replication at 24hpi in RIG-I+/+ and RIG-I-/- MEFs. Cells were transfected with si-HDAC6 and si-control for 36 h, followed infection with VSV-GFP (MOI = 1). bar, 100µm. E. HDAC6+/+, HDAC6-/- MEFs, and HDAC6-/- MEFs reconstituted with HDAC6 wild-type were subjected to immunoprecipitation using an anti-RIG-I antibody and immunoblotted with anti-acetyl-lysine and anti-RIG-I antibodies. Acetyl-tubulin was measured in total lysates to determine HDAC6 deacetylase activity. F. Whole cell lysates from HDAC6+/+ and HDAC6-/- MEFs were prepared and used in dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with anti-RIG-I, anti-HDAC6, and anti-β-actin antibodies. Intensity of pull-downed RIG-I was quantified. G. Whole cell lysates from HDAC6-/- MEFs and HDAC6-/- MEFs reconstituted with HDAC6 wild-type and HDAC6-CDM were prepared and used in dsRNA pull-down assays. Samples were analyzed by western blotting with anti-RIG-I and anti-HDAC6 antibodies. Intensity of pull-downed RIG-I was quantified.
Fig.Figure 6Cardiac insufficiency leads to cerebral hypoperfusion, which is in line with a hippocampal gene-expression response linked to oxidative, and ER-stress. It is likely that other factors in addition to hypoperfusion, such as circulating factors that may affect brain function, also play a role. Our data suggest that oxidative and ER-stress drive reduced expression of genes important for memory function, which involves reduced neuronal H3K4me3 representing loss of euchromatin. Other epigenetic changes are likely to further contribute to this effect. Administration of the HDAC inhibitor Vorinostat partially increases the expression of memory-related genes but also decreases the expression of genes linked to oxidative and ER-stress via the induction of a compensatory microRNA cluster.
Figure 7(A) rISG20-Fc treatment alleviates maternal viral burden. Note that rISG20-Fc treatment significantly decreased the maternal serum viral titers. Data represent as mean ± SEM; n=3 mice for control PBS group, and n=4 mice for treatment groups; *p < 0.05 by Student's t-test. (B) rISG20-Fc treatment reduces ZIKV titers in the fetal brain. Although there was no difference of ZIKV titer in the placenta, at the fetal side, there was a significant decrease of viral titer in the fetal brain, suggesting rISG20-Fc can block viral transmission from placenta to fetus. Data represent as mean ± SEM; n=4 for each group, and 3-4 placentas/fetal brains from every mouse were analyzed; *p < 0.05 by Student's t-test. ( (C) rISG20-Fc treatment decreases Eotaxin significantly in maternal circulation. Data represent as mean ± SEM; n=2 for PBS control group, and n=3 for every treatment group; *p < 0.05 by One-way ANOVA; difference not significant for IL-9 and IFNγ by One-way ANOVA. (D) Representative hematoxylin and eosin staining of the mouse placenta on E14.5. Labyrinth layers were marked with a solid line. Note the major alteration on placenta structure characterized by multifocal loss of tissue architecture (necrosis) and the defective blood vessel formation of labyrinth in ZIKV+PBS group, and rISG20-Fc treatment improves vascularity and decreases decidual edema and cellular fragmentation at the labyrinth. Scale bar=580μm (upper images); Scale bar=230μm (bottom images).
Figure 4A and C. Root mean square deviation (RMSD) analysis of the movement of (A) αC-helix and (C) DFG motif in the active and inactive conformation of ABL1 kinase with R351W mutation (orange) compared to the wild type (WT) kinase (black). B and D. Time-lapse images show movement within the αC-helix (B) and DFG motif (D) of the inactive structure of the WT (black outline) and R351W (orange outline) constructs. E. left, western blot analysis of transient overexpression of wild type ABL1 (WT) and ABL1-R351W, compared to empty vector control (EV) in HEK293T cells. Right, quantification of p-CRKL signal based on densitometry analysis of blots from 6 independent experiments. Error bars represent ±S.D., n=6, *P=0.0004 by two-tailed unpaired Student's t-test. F. Overexpression of mutant ABL1 constructs with mutations identified in selected primary lung tumors (Ding et al, 2008) in H157 cells. Values indicate quantified band intensity for p-CRKL, relative to α-tubulin and HA-tagged ABL1.