EMBO/sd-character-level-ner huggingface dataset:
Figure 1A, Schematic outline of plasma membrane D4H labeling. CM and LPDS indicate complete medium (10 % FBS in DMEM) and 5 % LPDS in DMEM, respectively. B, After 24 h LDL depletion in 5 % LPDS, cells were loaded with 50 μg/ml LDL for the indicated times. Plasma membrane cholesterol was labeled with D4H and imaged with widefield epifluorescence microscopy. C, Analysis of B. Mean ± SD, n = 15 fields of cells as in B pooled from 2 independent experiments. D, Representative FA lifetime in cells treated without or with 50 μg/ml LDL (-LDL or +LDL) for 1-2 h after 24 h in 5 % LPDS. Time-lapse imaging of GFP-paxillin dynamics in cells was acquired by TIRF microscopy and maximum intensity during FA lifetime was normalized to 1. E, Analysis of FA assembly and disassembly rate of cells incubated +/-LDL as in D. Where indicated, 10 μM PF228 was added after 30 min of LDL loading. Assembly rate: n = 290 FAs (-LDL); 410 (+LDL); 192 (+LDL+PF228), disassembly rate: n = 397 (-LDL); 502 (+LDL); 214 (+LDL+PF228) pooled from 4 independent experiments. The Tukey box plots represent the median and 25th and 75th percentiles (interquartile range). The whiskers show the hightest and lowest observations. Non-parametric Kruskal-Wallis analysis of variance with Bonferroni error correction. F, Representative immunoblot and quantification of pFAK levels in 5% LPDS (-LDL) and upon increasing LDL loading times. Mean ± SD, n = 3, paired student's t-test (*p < 0.05). G, Representative confocal images of NPC1, internalized integrin β1 and pFAK localization. Stably NPC1-GFP expressing cells were loaded with 2 μg/mL integrin β1 antibody for 30 min before fixation and stained with pFAK antibody followed by secondary antibodies. Dashed line indicates the cell edge. H, Quantification of G using Mander's colocalization for the indicated markers. Mean ± SD, n = 5 cells. Cells were treated with 5 % LPDS for 1 day and then without (-) or with (+) 50 μg/ml LDL for 1h.
Accumulation of NBR1 and its increased enrichment in the insoluble protein fraction in the atg7 mutant under heat stress.Figure 11Leaf tissues from wild type (WT) and atg7 expressing NBR1-TAP were collected at indicated hours (h) under 45°C and prepared for soluble and insoluble proteins as described in Materials and Methods. Proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and probed with a peroxidase-conjugated anti-peroxidase antibody for detection of NBR1-TAP. Protein samples prepared from non-transgenic Col-0 wild-type plants were included as controls.
Metabolomic fingerprints of primary mitochondrial diseases A - D Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of IOSCA (A), MIRAS (B), PEO (C), and MELAS/MIDD (D).Figure 1Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of IOSCA (A) Data information Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3 CDCA, chenodeoxycholic acid Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of MIRAS (B) Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3 CDCA, chenodeoxycholic acid Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of PEO (C) Data information: Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3; CDCA, chenodeoxycholic acid; GABA, γ-aminobutyric acid; OH-Kyn, 3-hydroxy-DL-kynurenine A - D Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of MELAS/MIDD (D). Data information: Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3 HIAA, 5-Hydroxyindole-3-acetic acid; SDMA, symmetric dimethylarginine; TCA, taurocholic acid.
a, Hierarchical cluster analysis of predicted REST targets, based on the presence of the canonical RE1 motif, shows relatively high expression in young adults (red) and lower expression (blue) in the ageing population.f1a, Hierarchical cluster analysis of predicted REST targets, based on the presence of the canonical RE1 motif, shows relatively high expression in young adults (red) and lower expression (blue) in the ageing population. Each lane represents an individual prefrontal cortical brain sample. b, qRT-PCR shows age-dependent induction of REST mRNA in the PFC. Values represent the mean ± s.d., n = 3. Shown are P values indicating significance for the mean of aged (71-95 years) versus young (24-29 years) cases for each primer. c, Age-dependent increase in total REST protein level in the PFC of controls but not AD patients. REST levels were also measured in isolated nuclear (Nuc REST; lower right panel, upper blot) and cytoplasmic (Cyt REST; lower right panel, lower blot) fractions. Each lane represents an individual case. Young, n = 12; Aged, n = 15; AD, n = 10. d, Confocal immunofluorescence labelling for REST (green), the neuronal marker MAP2 (red) and DNA (DAPI, blue) in the PFC of young adult, aged and AD cases. Scale bar, 25 µm. e, Quantitative analysis of nuclearREST levels by in situ imaging (left panel: Young, n = 11; Aged, n = 77; AD, n = 72; MCI = 11) or FACS analysis of isolated PFC neuronalnuclei (right panel: Young, n = 11; Aged, n = 22; AD, n = 11; MCI n = 12). For c and e, values are expressed as fold change relative to the young adult group, and represent the mean ± s.e.m. *P 0.05, **P 0.01, ***P 0.001 by Student's unpaired t-test. f, ChIP analysis shows induction of REST-RE1 site binding in normal ageing PFC neurons but not in AD.
Fig.Figure 3(A) A diagram showing Gpr56 WT, null and S4 transcripts. Solid boxes indicating exons that are transcribed. Arrowheads indicate the translation start sites. (B) Gpr56 transcript levels in isolated microglia from Gpr56 S4 mice. (C) A table showing the expression status of Gpr56 WT and S4 variants in microglia of different transgenic mice. (D) Representative images of Nissl staining of Gpr56 null and Gpr56 S4 E16.5 neocortex. Arrows indicate cortical ectopias that are shown in insets. (E) Quantification of ectopia size per section. (F) The distribution of ectopia from rostral to caudal cortex. (G). Myelin basic protein (MBP) staining of corpus callosum in P28 controls, Gpr56 null and Gpr56 S4 mice. (H) Quantification of MBP intensity in corpus callosum. (I) Gpr56 transcript levels in WT microglia at P5 and P14. (J) An overview of vGlut2 and Homer1 staining in dLGN at P10. The yellow outline indicates the dLGN core, and the dotted boxes show where synapses are quantified. K) Representative images of vGlut2/Homer1 staining in the dLGN of control, Gpr56 null, and Gpr56 S4 brains at P10. Arrows pointing to the enlarged synapse in the insets. L) Relative vGlut2/Homer1 synapse density in dLGN. N=10 (Ctrl), N=6 (Gpr56 null), N=3 (Gpr56 S4). (M) A schematic representation of the in vivo engulfment assay. CTB are injected into both eyes at P4, and anterogradely trace RGC projections to the dLGN. After 24 hours, the brains are collected and analyzed at P5. (N) Representative images and surface rendered microglia (green) in which CTB+(red) RGC inputs were engulfed. (O) Quantification of the percentage of engulfed RGC inputs in controls, Gpr56 null and Gpr56 S4 microglia.
Reduced Aβ load and ameliorated cognitive function in Mgat3−/− mouse brain.Figure 4Amounts of Aβ40 or Aβ42 in the TBS-soluble or Gu-HCl-extractable fraction from (A) 3-month-old or (B) 12-month-old hAPP/Mgat3+/+, hAPP/Mgat3+/−, or hAPP/Mgat3−/− brains (n = 5). P = 0.015 for TBS Aβ40, P = 0.034 for TBS Aβ42, P = 0.001 and 0.012 for Gn-HCl Aβ40, P = 0.024 for Gn-HCl Aβ42 in (A), P = 0.040 for Gn-HCl Aβ40, P = 0.047 for Gn-HCl Aβ42 in (B). Amounts of Aβ40 or Aβ42 in the TBS-soluble or Gu-HCl-extractable fraction from 3- to 4-month-old Mgat3+/+ or Mgat3−/− brains (n = 7-8). An outlier value was rejected by the Smirnov-Grubbs' test (P < 0.05). P = 0.045 for Gn-HCl Aβ40. Immunostaining of Aβ plaques in 12-month-old mouse brain (left). Scale bar, 300 μm. The number of FSB-stained Aβ plaques in brain sections prepared from 12-month-old male mice was quantified (right) (n = 6). P = 0.004 for hippocampus, P = 0.0003 for cortex. The Y-maze test was performed using 12-month-old male Mgat3+/+, Mgat3−/−, hAPP/Mgat3+/+, or hAPP/Mgat3−/− mice (n = 8-10). **P = 0.004 for Mgat3+/+ versus hAPP/Mgat3+/+, *P = 0.022 for hAPP/Mgat3+/+ versus hAPP/Mgat3−/−.
Figure 3A. Cells from mock-organoids display a healthy nucleus labeled by DAPI (blue) (i). SARS-CoV-2-positive cells (green) display condensed (middle panel, ii) and fragmented DNA (bottom panel, iii, arrows). At least 75 cells from two (n=2) independent batches of organoids were examined. Figures display scale bars. B. Compare to mock organoids (i) SARS-CoV-2-exposed organoids (ii) display increased TUNEL-positive cells (displayed as inverted LUT) at the cortical plate that is specified by TUJ-1 (magenta). At least four organoids from two (n=2) independent batches of organoids were examined. Figures display scale bars. The bar diagram below quantifies the frequencies of TUNEL-positive cells between mock and SARS-CoV-2-exposed organoids. Four organoids from two (n=2) independent batches were examined. Unpaired t test, *P<0.05. Data presented as mean + SD. C. Most of the SARS-CoV-2-positive cells (AB4, green) are TUNEL-positive (i) and some of the SARS-CoV-2-positive cells are caspase-positive (ii). Caspase-positive cells in SARS-CoV-2-exposed organoids display pT231Tau localization at the cell soma specified by AT-180 (ii), which are not observed in in mock organoids (iii). Arrowheads point SARS-CoV-2-positive cells (AB4, green) that are also positive for TUNNEL (red), caspase 3 (yellow) and Tau AT-180 (magenta). Figures display scale bars. Bar diagrams at right quantifies proportions of TUNEL and caspase-positive cells among SARS-CoV-2-positive cells. The second graph below quantifies the proportions of pT231Tau-positive cells among caspase positive cells between control and virus exposed groups. At least 400 cells from 4 organoids and two independent (n=2) batches were examined. Unpaired t test, **P<0.01. Data presented as mean + SEM. TUNEL-positive cells in control un-exposed organoids do not contain pT231Tau (iii). Figures display scale bars. Bar diagrams at right quantifies proportions of pT231Tau-positive cells among TUNNEL-positive cells between control and virus exposed groups. At least 350 cells from 4 organoids and two independent (n=2) batches were examined. Unpaired t test, ***P<0.001. Data presented as mean + SEM.
Immuno-EM of p62-positive structures in Vps24- and Tsg101-depleted cells.Figure 6.Immuno-EM of p62-positive structures in Vps24- and Tsg101-depleted cells. Cryosections of HeLa cells transfected with Vps24 (A, C, and E) or Tsg101 (B, D, and F) siRNA were incubated with antibodies against p62 (15-nm protein A gold, 10-nm in D). Membrane-free p62-positive aggregates (C and D) or p62-positive structures contained within dense clusters of vesicular-tubular elements (A and B) were detected. Vesicles with early endosomal morphology (EE) or multivesicular appearance (MVB) associated with these clusters. We also observed p62 labeling in electron-dense structures sequestered within amphisomes in cell depleted of Vps24 (E) or Tsg101 (F). The amphisomes typically consist of an electron-dense p62 positive body (E and F, arrowhead), and a multivesicular endosomal structure (E and F, arrow). Bars, 200 nm.
Wide-spread expression of proliferation-associated genes in diencephalic astrocytesA, B t-Distributed stochastic neighbor embedding visualization of ACSA-2-MACSed cell populations of representative well-known proliferation genes (A; numbers reflect scaled log normalized read counts for each cell, Dataset EV3 for A, B) and associated violin plot (B) showing distribution of proliferative genes across all clusters (cluster color coding and annotation see in Fig.Figure 5A, B t-Distributed stochastic neighbor embedding visualization of ACSA-2-MACSed cell populations of representative well-known proliferation genes (A; numbers reflect scaled log normalized read counts for each cell, and associated violin plot (B) showing distribution of proliferative genes across all clusters (cluster color coding as and value per each single cell (A). C, D Velocity analysis based on differences between spliced and unspliced transcripts for all cell clusters (C) and astrocyte clusters (D).
Figure 8Figure 8. Overexpression of the CHCHD10 mutant alleles in HeLa cells leads to inhibition of apoptotic cell death. Transfections were performed with empty vector (EV) or vectors encoding either wild-type CHCHD10-FLAG (WT) or mutant CHCHD10-FLAG (P34S and S59L). A. Western blot on HeLa cells extracts using antibodies against FLAG or Hsp90. B-D. HeLa cells transiently expressing the WT or mutated forms of CHCHD10-FLAG (P34S or S59L) were treated with 1 μM Actinomycine D (ActD) for 4, 6 or 8 h with measurement of Annexin V/DAPI staining (B), DEVD-ase activity (C) from three independent experiments. Differences between the mutated and non mutated alleles were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01) or very significant (**:0.01>p>0.001). P34S versus WT: **: p=0.0055 (4h), *: p=0.0103 (8h). S59L versus WT: *: p=0.0304 (4h), *: p=0.0129 (8h). D. Mitochondrial outer membrane permeabilization was determined by western blot by assessing the degradation of Smac. Hsp90 was used as a loading control.
Figure 6Relative quadriceps (Quad) mRNA expression of the ISR components Atf4, Atf5, Atf6 and Chop (WT day n=9, WT night n=5, TG day n=20, TG night n=12). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm). Representative immunoblots of ISR component eIF2α (B) and quantification of phospho-eIF2α (p-eIF2aSer51) relative protein expression (C) in skeletal muscle (Gastroc, WT day n=4, WT night n=5, TG day n=9, TG night n=9). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm). Relative mRNA expression of Rev-erba (D) and Gdf15 (E) in quadriceps, liver and eWAT (WT day n=9, WT night n=5, TG day n=20, TG night n=12). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm). Skeletal muscle (Quad) GDF15 protein content normalized to total protein content (WT day n=4, WT night n=5, TG day n=14, TG night n=12) Circulating GDF15 plasma levels (WT day n=9, WT night n=5, TG day n=18, TG night n=12). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm). Graphical summary showing muscle mitochondrial stress induced secretion and diurnal variation of GDF15 to drive a daytime-restricted anorexia, affecting in turn metabolic flexibility, adiposity, WAT browning and systemic insulin sensitivity. Parts of this cartoon were created using Servier Medical Art (http://smart.servier.com). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm).
AR regulation of gene networks in prostate cancerImmunohistochemical (IHC) detection of AR in four malignant prostate tumors and two paired NATs.Figure 7A Immunohistochemistry (IHC) detection of AR in four malignant prostatetumors and two paired NATs. Representative microphotographs of HE staining showed normal prostatic gland (NATs) and poorly differentiated adenocarcinoma cells. IHC analysis indicated heterogeneous AR expression among patients. Magnification, 40×. N1a and M3a were collected from the same patient ((Supplementary Table S5). N2b and M4b were gathered from another patient ((Supplementary Table S5). B Tissue ARChIP‐exo raw signal intensity showing 1,715 enhanced AR binding locations in malignant prostatetumors (M) compared to NATs (N). ChIP‐exo data of LNCaP (+DHT) is plotted around the same locations. C Pathways and networks constructed using genes within 50 kb of AR binding locations enhanced in prostate tumors. Nodes and corresponding pathways are extracted according to interaction annotation. Literature‐based network is configured as PROSTATIC NEOPLASMS (Genomatix Mining). D Oncomine analysis showing median rank for genes in the PROSTATIC NEOPLASMS network across 16 analyses of overexpression in prostate cancer (PCa) versus normal. The right column represents fold change in gene expression detected by RNA‐seq in the LNCaP cell line after DHT treatment (Log2 FC DHT/Vehicle, P 0.01). E Survival curves were generated based on Kaplan-Meier analysis. For each of the four clinical datasets, up to one‐third of genes in the PROSTATIC NEOPLASMS network was selected based on gene expression level. These genes were then used to stratify patients. The P‐values were calculated by log‐rank test.
Irgm2 cooperates with GATE16 to dampen Gram-negative bacteria-induced non-canonical inflammasome activation (A) GFP-Trap coupled to mass spectrometry strategy used.Figure 3(A) GFP-Trap coupled to mass spectrometry strategy used. The volcano plot represents 3 independent combined experiments. Threshold selection of enriched proteins (red dots) in Irgm2-GFP fraction was set at 2 fold enrichment (x axis) and p-value <0.05 (y axis). Blacks and Grey dots indicate proteins that did not reach a p-value <0.05 using t-test with bonferroni correction. Labelled proteins represent the top 6 enriched proteins in the Irgm2 fraction. (B) Green Florescent Protein (GFP)-Trap assay of the presence of Gate16 in Irgm2-GFP-enriched fraction from the lysates of IFNγ-primed iIrgm2-/- BMDMs complemented with lentiviral constructs coding for a fusion of Irgm2 withGFP (Irgm2-GFP) or GFP alone. Arrows show the presence or not of Gate16, Irgm2-GFP and GAPDH in the Irgm2-GFP-enriched fractions, flow-through and total cell lysates. * means non-specific band (C) LDH and IL-1β release from siRNA-treated WT BMDMs, and then exposed to 2.5 µg/2.105 cells of OMVs for 16 hours. (D) LDH and IL-1β release from WT, Irgm2-/- and Casp11-/- BMDMs silenced for Gate16 and treated for 16 hours with 2,5µg/2.105 cells of OMVs. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences. (E) Western blot examination of caspase-11 and processed caspase-1 (p20) and gasdermin-D (p30) in supernatants and pro-caspase-1 (p45), pro-gasdermin-D (p55), pro-caspase-11, Gate16 and GAPDH in cell lysates of Gate16-silenced WT, Irgm2-/- and GBPChr3-/- BMDMs exposed to 2,5.105µg/2.105 cells of OMVs for 16 hours. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences. (F) Representative confocal fluorescence microscopy images and associated quantifications of caspase-11-C254G-GFP (green) recruitment to S.Tm-mCherry (orgA-, red, MOI 10) in IFNγ-primed iWT BMDMs silenced for Gate16 after 4 and 8 hours of infection. Nucleus (blue) was stained with Hoechst; scale bar 5µm. For quantifications, the percentage of bacteria positive for caspase-11-C254G-GFP was determined by combining the bacterial counts from n=3 independent experiments and expressed as mean ± SEM. **p ≤ 0.01, ***p ≤ 0.001 for the indicated comparisons using t-test with bonferroni correction. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.
Figure 7Diagram for ablation of CX3CR1+ macrophages using CX3CR1-DTR (DTR) mice. DT, diphtheria toxin. Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and DTR mice (n = 5 mice/group). Comparison of mRNA levels of VEGF-C in the whole tissue of jejunum and ileum from WT and DTR mice (n = 5 mice/group). Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and DTR mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm. Images and comparisons of number of Prox1+ LECs within initial 100 μm portion (upper) and VEGFR3 expression (lower, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from WT and DTR mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm. Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between WT and DTR mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.
In the absence of reperfusion, genetic inhibition of PI3KC2β does not affect inflammatory cytokine profile and infarct volume.Figure 3A Gene expression of TNFα (n=8-10 mice for WT and n=6 mice for C2βD1212A/D1212A), IL-1β (n=8 mice for WT and n=10-12 mice for C2βD1212A/D1212A), IL-6 (n=8 mice for WT and n=10-12 mice for C2βD1212A/D1212A) and VCAM-1 (n=8-10 mice for WT and n=10 mice for C2βD1212A/D1212A) after 1 hour of ischemia without reperfusion (right panel). Gene expression of IL-6 (n=10-12 mice for WT and n=8 mice for C2βD1212A/D1212A) and VCAM-1 (n=12 mice for WT and n=10mice for C2βD1212A/D1212A) after 1 hour of ischemia followed by 4 hours of reperfusion (bottom, left panel) on WT and C2βD1212A/D1212A mice. The mRNA levels are given as the fold increase normalized to PGK1 relative to the healthy contralateral hemisphere of WT mice. Data represent mean ± SEM (***p<0.001, **p<0.01, *p<0.05, Mann-Whitney test or unpaired t-test, as accordance). B Representative photographs of T2-weighted MRI of WT and C2βD1212A/D1212A mice and graph quantification of ischemic lesion after 24 hours permanent MCAO (electrocoagulation model). Data represent mean ± SEM (n=9-10 mice per group; ns, unpaired t-test).
Figure 5RT-qPCR analysis of relative mRNA levels for four representative genes following OptoCAR-mediated T cell activation. OptoCAR‑T cell conjugates were activated in the dark (signaling competent) state for a period of 3 hours before light-mediated disruption of signaling (open circles) or maintained for a further 60 minutes in the dark (filled circles). The relative mRNA values were calculated by subtracting baseline value before scaling to mean output between 3-4 hours. Individual datasets are presented as gray lines, with bars showing mean ± SEM (n=3). Raw cRQ plots for all datasets are provided in Appendix Fig S2 and RQ values can be found in Dataset EV1. The datasets from (A) are expanded to show the effect of illumination on the relative mRNA level of designated genes. As above, datasets are normalized to the mean mRNA level for the dark state values over the 60-minute period. Bars show mean ± SEM (n=3). The ratio of relative mRNA in the light compared to dark state were calculated for each timepoint. An approximate half-life for mRNA decay is shown with dotted lines. Bars show mean ± SEM (n=3).