EMBO/sd-character-level-ner huggingface dataset:
A-YTHDF1-dependent manner A LC-MS/MS quantification of the m6A/A in mRNA of control, wild-type (WT)- and mutant (MUT)-ALKBH5 overexpressing cells.Figure 4A LC-MS/MS quantification of the m6A/A in mRNA of control, wild-type (WT)- and mutant (MUT)-ALKBH5 overexpressing cells. B Western blot analysis of ALKBH5 and TRAF4 in control, WT- and MUT-ALKBH5 overexpressing cells and quantification of protein levels normalized to β-actin expression. C Integrative genomics viewer (IGV) plots of m6A peaks at TRAF4 mRNAs. The y-axis shows sequence read number, blue boxes represent exons, and blue lines represent introns. D MeRIP-qPCR analysis of m6A levels of TRAF4 mRNA in control and ALKBH5-depleted cells. E MeRIP-qPCR analysis of m6A levels of TRAF4 mRNA in in control, WT and MUT-ALKBH5 overexpressing cells. F Left panel: Schematic diagram of dual luciferase reporter constructs. Right panel: Relative luciferase activity of WT or MUT (A-to-T mutation) TRAF4 3ʹUTR luciferase reporter in control, WT and MUT-ALKBH5 overexpressing cells. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. G Western blot analysis of TRAF4 protein levels in control and YTHDF1 overexpressing cells and quantification of protein levels normalized to β-actin expression. H Polysome profiling assays. The fractionation of lysates from 3T3-L1 cells transfected with or without YTHDF1 plasmid is shown on the top. RNAs in different ribosome fractions were extracted and subjected to qPCR analysis. I RIP-qPCR analysis of the interaction of TRAF4 with FLAG in cells overexpressing FLAG-YTHDF1. Enrichment of TRAF4 with FLAG was measured by qPCR and normalized to input. Relative luciferase activity of WT or MUT TRAF4-3ʹUTR luciferase reporter in cells transfected with control or YTHDF1 plasmid. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. Western blot analysis of YTHDF1, ALKBH5 and TRAF4 in control, ALKBH5-depleted and ALKBH5 + YTHDF1 depleted cells and quantification of protein levels normalized to β-actin expression. L Oil Red O staining of control, ALKBH5-depleted and ALKBH5+YTHDF1 depleted cells on day 8 of differentiation. M qPCR analysis of PPARγ, C/EBPα and FABP4 expression in cell β-Actin was used as an internal control.
Figure 2 (A) Absolute cell counts from lungs of sub-lethally influenza virus infected mice following analysis by flow cytometry. After gating out alveolar macrophages by forward and side scatter, immune cells are defined as follows: Neutrophil (Ly6G+CD11b+SiglecF-Ly6Cint), Eosinophil (SiglecF+CD11c-/+F4/80-CD64-), Monocyte (Ly6G-CD11b+MHCII-SiglecF-), Natural killer cell (NK, B220-/intNK1.1+), B cell (B220hiNK1.1-), CD8+ T cell (TCRβ+CD8+), CD4+ T cell (TCRβ+CD4+) Data information: Data are representative of three independent experiments with 3-4 mice per group and represented as mean ± S.E.M. Statistical significance in (A) was determined with unpaired student's T-test. n.s.= not significant  (B) Flow cytometry plots of neutrophils and eosinophils in lungs at days 9 and 15 post sub-lethal influenza virus infection. Numbers in plots indicate frequency of gated immune cells in each throughput Data information: Data are representative of three independent experiments with 3-4 mice per group and represented as mean ± S.E.M
Figure 1A The scheme of construction of a diploid fertilized oocyte using a primary spermatocyte and a GV-stage oocyte. The chromosomes of the spermatocyte and the oocyte are intermingled at MI to form a single chromosomal mass. B Spermatocyte chromosomes that underwent condensation (right) within a MII oocyte (MII chromosome mass, left). In this study, spermatocytes picked up for injection had tetrad chromosomes ready for condensation. C Chromosomal analysis of MII oocytes that had been injected with primary spermatocytes. In the spermatocyte-injected groups, normality was improved by reducing the ooplasm mass (*P < 0.005 by Fisher's exact probability test). Arrows in the right figure indicate prematurely separated chromatids. The numbers of oocytes observed are indicated on the top of the bars.
a, Correlative light and electron microscopy of HeLa/GFP–LC3 cell infected with SIN/mCherry.f1a, Correlative light and electron microscopy of HeLa/GFP-LC3 cell infected with SIN/mCherry.capsid virus. Top left, deconvolved image of red and green fluorescence channels; middle, three-dimensional surface reconstruction of red and green channels; right, yellow (red + green) colocalization channel. Arrows denote yellow puncta that correspond to '1' and '2' in electron microscopy images below. Bottom left, electron microscopy of identical cell; middle and right, high magnification images of insets '1' and '2'. Scale bars, left, 10 µm; middle and right, 200 nm. b, Ranked distribution of median Z-scores for each siRNA pool in primary colocalization (virophagy) screen. Red, decreased colocalization; green, increased colocalization; blue, insufficient numbers of green or red puncta per cell or total number of cells per well for analysis. c, Maps of protein interactions in enriched network modules (see Supplementary Fig. 3b). d, Association of siRNA hits with autophagy network.
Figure 3(A) Representative hematoxylin and eosin (HE) staining of ileum. Paneth cells with typical eosinophilic granules (black arrows) on HE-stained sections at the base of crypts in Villin-Cre and COPS8-lox alleles expressing (COPS8fl/fl ), but not COPS8 knockout (KO) (COPS8ΔIEC) epithelium. Scale bar 50 μm, n=7 biological replicate per genotype. (B) The granule protein, lysozyme, was examined by immunofluorescence (Red) and counted in the ileum of COPS8fl/fl and COPS8ΔIEC mice (scale bar 100 μm). The column graph represents number of lysozyme+ cells presented as mean ± SEM from seven biological replicates. **P<0.01 using Student's t-test. (C) Transmission electron microscopy (TEM) of crypts of COPS8fl/fl and COPS8ΔIEC mice. The base of the crypt in COPS8ΔIEC mice is occupied by poorly differentiated columnar epithelial cells that lack secretory granules, rudimentary electron-dense granules (black arrows), granules in the lumen (blue arrows) and have a contracted endoplasmic reticulum (ER) (white arrows). Scale bar 5μm, data are from seven biological replicates per genotype. (D) Goblet cells were stained by Alcian blue and counted in COPS8fl/fl and COPS8ΔIEC epithelia of duodenum and jejunum (scale bar 100 μm). The column graph represents number of Alcian blue+ cells presented as mean ± SEM from seven biological replicates. *P<0.05 using Student's t-test. (E) The marker for enteroendocrine cells (between two dashed line), chromogranin, was detected by immunofluorescence and counted in the ileum of COPS8fl/fl and COPS8ΔIEC mice (scale bar 100 μm). The column graph represents number of chromogranin A+ cells per crypt-villus presented as mean ± SEM from seven biological replicates. *P<0.05 using Student's t-test. (F) RNA sequencing-based measurements of transcripts comprising antimicrobial peptides (AMPs)-related genes in crypts isolated from COPS8fl/fl and COPS8ΔIEC mice. Representative data from three biological replicates per genotype. (G-H) Real-time quantitative reverse transcription Polymerase chain reaction (PCR) (qRT-PCR) analysis of the expression of genes encoding AMPs in the ileum (G) and colon (H) in COPS8fl/fl and COPS8ΔIEC mice. Error bars indicate the mean ± SD from three biological replicates. *P<0.05, **P<0.01, ***P<0.001 using Student's t-test.
Figure 6(A) Representative confocal imaging of localization of Lamp1-mcherry and LysoTracker Green in wild type (w1118), NimB4 sk2 and draper△5 macrophages (live imaging). The arrows indicate the colocalization (w1118) or the clustering (NimB4sk2 and draper△5) of Lamp1-mcherry and LysoTracker Green signals. Scale bar: 10 μm. (B) Quantification of the colocalization of Lamp1-mcherry with LysoTracker Green, as measured by Pearson's correlation coefficient between the two signals. Values from at least five independent experiments are represented as mean ± SD (* P<0.05 by ANOVA test followed by post hoc Dunnett's multiple comparison tests). (C) Ex vivo phagocytosis assay using apoptotic cells labelled with pHrodo™ Red. Wild type (w1118), NimB4sk2 and draper△5 macrophages from L3 wandering larvae were incubated with pHrodo™ Red apoptotic cells for 60 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant). (D) Ex vivo phagocytosis assay using pHrodoTM Red S.aureus BioparticlesTM conjugates. Wild type, NimB4sk2 and NimC11, eater1 Hml∆-Gal4>UAS-GFP macrophages from L3 wandering larvae were incubated with pHrodoTM Red S. aureus BioparticlesTM for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).
Figure 2(A) Western blotting analysis confirmed HMCES knockout in 293A cells (293A HMCES KO). Blots also included knockout cells reconstituted with WT SFB- HMCES. (B, C) Colony-forming capacity of HMCES KO cells was compared to that of control wild-type 293A cells. (C) Box plot showing colony forming efficiencies of 293A wild-type control and HMCES KO cells. Box limits represent 25th and 75th percentile, centerline shows the median and whiskers extend minimum to maximum. Data include more than three independent repeats performed in duplicates. Student's t-test were performed for statistical analysis (****, p<0.0001). (D) Colonogenic survival assay of 293A wild-type control and HMCES KO cells treated with various concentrations of hydroxyurea (HU) was shown. Seeding density with respect to control was indicated below each image. The images shown are representative of three biological repeats, each performed in duplicate. (E) Quantification showing survival fraction of wild-type control (WT) and HMCES KO cells upon HU treatment. Data are presented as mean±s.e.m (n=3, each performed in duplicates) and student's t-test were performed for statistical analysis (ns, not significant; *, p<0.05). (F) Quantification of colonogenic survival assay upon exposure of WT and HMCES KO cells to ionizing radiation (IR). Data are presented as mean±s.e.m. Quantifications are from three biological repeats performed in duplicates. Student's t-test was performed for statistical analysis (*, p<0.05; **, p<0.01). (G, H) Sensitivity assay and quantification of surviving WT control and HMCES KO cells determined by colony-forming assay upon exposure to H2O2. Experiments were repeated three independent times, each time performed in duplicates and data are presented as mean±s.e.m. Statistical significance for the corresponding experiment was analyzed by student's t-test (*, p<0.05). (I) Bar graph showing sensitivity of WT, HMCES KO and stably reconstituted WT cells towards H2O2 treatment. Experiment was repeated two independent times with technical repeats.
Fig.Figure 1(A) Immunoblot of HIV-1 GFP virus particles (2×1011 genomes) produced with lopinavir (LPV, 0-100 nM) detecting p24 antibody. Titration of LPV-treated HIV-1 GFP viruses on PMA-treated THP-1 shSAMHD1 cells. Mean ± SD, n=3. Titration of LPV-treated HIV-1 GFP viruses on PMA-treated U87 cells. Mean ± SD, n=3. ISG qPCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). ISG qPCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). ISG qPCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). G) CXCL-10 protein in cell supernatants from D-F (ELISA). (H) ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.2 U RT/ml 30 nM LPV-treated HIV-1 GFP in the presence of DMSO vehicle or 2 μM ruxolitinib. A control was stimulated with 1 ng/ml IFNβ. (I) RT products from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.3 U RT/ml LPV-treated HIV-1 GFP viruses. (J) Immunoblot of HIV-1 R9 BaL virus particles (2×1011 genomes) produced with LPV (0-100 nM) detecting p24. (K, L) ISG qRT-PCR (K) and infection data (L) from primary monocyte-derived macrophages (MDM) infected for 24 h with LPV-treated HIV-1 R9 BaL viruses (0.2 U RT/ml). Data are collated from two donors (represented by circles and squares), n=3. Horizontal line represents the median. For experiments in which the virus dose used was normalised by RT activity, the number of genome copies was also measured by qPCR of virus. This gave dose equivalents of within 2-3 fold of RT equivalents.
a) Immunoblots of rat liver lysosomes incubated with WT or G/S LRRK2 alone or with 1 mM GTP and/or GAPDH.figf5(a) Immunoblots of rat liver lysosomes incubated with WT or G/S LRRK2 alone or with 1 mM GTP and/or GAPDH. Duplicate samples are shown. Bottom: quantification of lysosome-bound LRRK2 expressed as a multiple of the amount bound without additions (n = 6 independent experiments). (b) Effect of kinase inhibitor on WT, G/S, or the kinase-dead mutant D/A LRRK2 incubated with rat liver lysosomes in presence or absence of GAPDH (5 μg). Right: quantification expressed as a multiple of the association without additions (n = 3-5 independent experiments). (c,d) Transient transfection of HEK293 cells with the myc- or GFP-tagged LRRK2 constructs indicated at top left in c. Full, full length; M, KFERQ-like motifs (M1-M8). (c) Binding to GST-hsc70. Left, representative immunoblots; input lanes, one-tenth of input material. NT, non-transfected cells. Immunoblot for hsc70 is shown as loading control. Top right: quantification of LRRK2 bound to hsc70. Where indicated, samples were also incubated with RNase A to compete the KFERQ-mediated binding of hsc70 (n = 3 or 4 independent experiments). (d) Coimmunoprecipitation (IP) of hsc70 with anti-myc or anti-GFP in the same cells. Inputs (Inp), one-quarter of starting volume. (e) Binding or LRRK2 to rat liver lysosomes treated with the indicated concentrations of trypsin. LAMP-1 is shown to confirm the integrity of the lysosomes. (f) Co-IP of LAMP-2A with anti-LRRK2 in lysosomes incubated with WT or G/S LRRK2. Inp, one-quarter of starting sample; IgG, immunoglobulin G. Values: LAMP-2A recovered in the IP (IP L2A) corrected for the amount of LRRK2 pulled down and expressed as a multiple of WT value. (g,h) Duplicate LAMP-2A immunoblots of rat liver lysosomes incubated alone (−) or with WT or G/S LRRK2 (g) or with lysosomes from control (Ctr) or from WT-LRRK2 or G/S-LRRK2 transgenic mice (h) and subjected to blue native electrophoresis. Arrow, 700-kDa multimeric complex. All values are mean + s.e.m.; differences compared to none or untreated (*) or to WT or full-length protein (§) were significant at P 0.05; unpaired t-test in a and ANOVA and Bonferroni test in b,c. Full-length blots and gels in Supplementary Figure 12.
Figure 4A. Heatmap analysis of differentially expressed genes between SATB2 silencing H2S GSCs (shSATB2) and control H2S GSCs (shNT). Differentially expressed genes had a 1.8-fold or greater expression difference. Among differentially expressed genes, 160 are upregulated and 185 are downregulated. B. Gene ontology analysis of genes downregulated in SATB2 silencing GSCs compared with control GSCs. The most significantly down-regulated genes are associated with cell cycle progression and chromosome organization. p values were generated using the PANTHER tool (http://pantherdb.org/). C. qPCR analysis of FOXM1 and SATB2 mRNA expression in GSCs transduced with shNT or shSATB2 (n=3). D. Immunoblot analysis of FOXM1 and SATB2 expression in GSCs transduced with shNT or shSATB2. E. Immunoblot analysis of FOXM1 and SATB2 expression in tumor xenografts-derived from T3359 GSCs expressing shNT or shSATB2. F. Heatmap analysis of FOXM1 downstream targets between SATB2 silencing H2S GSCs (shSATB2) and control H2S GSCs (shNT) from microarray analysis. Differentially expressed genes had a 1.5-fold or greater expression difference. G. qPCR analysis of FOXM1 downstream targets in H2S GSCs transduced with shNT or shSATB2 (n=3).
Figure 2A Representative images of humanneurons cells stained with the mitochondrial membrane potential sensible fluorescent probe TMRM, the neuronal specific anti-NCAM antibody and the nuclear staining Hoechst. Left panel, basal conditions. Right panel, after addition of the mitochondrial uncoupler FCCP. Scale bar 20 μm. Plot showing the quantification of TMRM fluorescence signal from NCAM+neurons. Data presented as means + SEM of three independent experiments (unpaired, two-tailed t-test). B Representative images of ultrastructural analysis of fixed neurons examined with electron microscope. PANK2 panel represents PKAN neuronal cells overexpressing PANK2 protein. Scale bar 500nm. Mitochondrial size was measured at level of the larger diameter along the perpendicular axis for all the mitochondria in >30 field (200 mitochondria in total) for each sample (one-way ANOVA). C OCR measurements of controls and each PKAN patients analyzed individually and D data obtained by individual analysis and plotted as grouped controls, PKAN patients and PKAN patients overexpressing PANK2. The plots show OCR normalization to cell number. OCR was measured in basal conditions, and after oligomycin and FCCP addition. Bars indicate means + SEM of three independent experiments (Two-way ANOVA).
Figure 4Representative microphotographs of TLR4 silenced and non-silenced MSCs primed with LPS and co-cultured with neutrophils. Immunostaining was performed with an antibody detecting NETs (DNA-histone, red). Of note, TLR4 silenced MSCs upon LPS priming cannot mount any adaptive response and fail to enhance NET formation (lower row, outer right panel) as compared to non-silenced, LPS primed MSCs co-cultured with PMA activated neutrophils (lower row, outer left panel). Neutrophils and DMSO served as a negative control (upper row, outer left panel), while neutrophils activated by the protein C kinase activator PMA served as a positive control depicting enhanced NET formation (upper row, middle panel). Co-culture with MSCs suppressed enhanced NET formation (upper row, outer left panel) induced by PMA. Scale bars: 50μm. Quantitative assessment of NET bound elastase employing a specific ELISA in identical experimental setting as described in Figure 1. Similarly, to immunostaining, a significant reduction of NET bound elastase was observed in TLR4 silenced LPS primed MSCs when co-cultured with activated neutrophils as opposed to high NET bound elastase in non-silenced LPS primed MSCs co-cultured with activated neutrophils. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, three biological replicates. High resolution scanning electron microscope analysis depicting enhanced NET formation (red arrows) expulsed from neutrophils (blue arrows) in the presence of LPS primed MSC co-cultured with PMA activated neutrophils (middle row, outer left panel) as compared to reduced NETs in non-primed MSCs co-cultured with activated neutrophils (middle row, outer right panel). Of note, TLR4 silenced LPS primed MSCs failed to activate neutrophils and NETs (lower row, outer left panel). TLR4 silenced and non-primed MSCs display reduced NETs in co-cultures with activated neutrophils (lower row, outer right panel), suggesting that PMA activation shares TLR4 signaling components. PMA activated neutrophils alone served as positive control with highly enhanced NETs (upper row, outer right panel) and DMSO treated neutrophils as negative controls (upper row, outer left panel). Red stars indicate neutrophil derived granules. Scale bars: 0.1µm
Changes with time in amounts of SH-EP, α-amylase, and VmPE-1 in cotyledons of V.Figure 1.Changes with time in amounts of SH-EP, α-amylase, and VmPE-1 in cotyledons of V. mungo seedlings (Attached cotyledons) or embryo axis-removed cotyledons of V. mungo seeds (Detached cotyledons). Attached or detached cotyledons were prepared as described in Materials and methods. 10 pairs of cotyledons were homogenized with 3 ml of 50 mM Tris-Cl (pH 7.4), and the homogenates were centrifuged at 15,000 g for 10 min. Supernatants (10 μl each) were analyzed by SDS-PAGE immunoblotting with anti-SH-EP, α-amylase, or VmPE-1 antibody.
Figure 5Mice bearing epithelial (red text) and mesenchymal (blue text) non-small cell lung cancer tumors were treated with vehicle control, volasertib (30 mg/kg per week intravenously), tepotinib (25 mg/kg per day orally), or the combination for 5 weeks to generate tumor growth curves of patient-derived xenografts (PDX; A) and cell line xenografts The percent change in tumor volume at the end of treatment (normalized to day zero) was calculated with each data point representing an individual mouse, and the bar is the mean value ± standard error of 10 mice for each group Tumor growth curve of mesenchymal PDX TC424 (top) and cell line xenograft Calu6 (bottom) upon treatment for 5 weeks and after cessation of treatment (recovery phase). Data are means ± standard error of the mean from five mice for each group. Kaplan-Meier survival curves for mesenchymal PDX TC424 (top) and cell line xenograft Calu6 (bottom) with death due to excessive tumor burden as the endpoint. Quantification of TUNEL-positive cells in paraffin-embedded xenograft tissue sections.
Figure 2DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (A) Representative T1-weighted image of a pregnant WT mouse and a selection of four different placentas (outlined by colored lines). DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (B) Representative SI dynamics of a single WT placenta (marked in A), following biotin-BSA-GdDTPA administered for 60 min. SI parameters of Initial Enhancement and Recovery are illustrated. DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (C) Representative SI dynamics of a single WT placenta (marked in A) over time. Scale bar, 2.5 mm. DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (D−G) Representative SI plots of Cdkn1a-/- (D), p53-/- (E), Cdkn2a-/- (F), and Cdkn2a-/-;p53-/- (G) placentas, compared to their WT or HET littermates. (H−I) Quantification of the SI Initial Enhancement (H) and SI Recovery (I) in WT, Cdkn1a-/-, p53-/-, Cdkn2a-/-, and Cdkn2a-/-;p53-/- placentas. MRI experiments were repeated at least three times for each murine genotype (n>=3 placental SI measurements from each genotype). Values are means +SEM; Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.
Structure–function studies identify critical molecular determinants of the anti-inflammatory bioactivity.Figure 6Dose-response curves showing the modulation of R837-induced (5 μg/ml; 18 h) IL-12 secretion by prior treatment of BMDCs with the indicated synthetic lipids for 1 h.Chemical structures and ΔEC50 values of the synthetic OxPL variant lipids analyzed in (A), presented relative to EC.