EMBO/sd-character-level-ner huggingface dataset:
Figure 3(A) Representative images of immunohistochemical staining of IL-1β, caspase-1, and GSDMD in the brains of indicated group. Scale bars, 50 μm. (B) Quantitative analysis for immunohistochemical staining. n = 6 mice per group. (C) Representative images of double-immunofluorescent stained for GFAP and GSDMD, for Iba-1 and GSDMD, and for NeuN and GSDMD in post-ischemic brains after tMCAO. (D) Representative images of double-immunofluorescent stained for Iba-1 and GSDMD, for Iba-1 and caspase-1, and for Iba-1 and IL-1β of sham+vehicle, MCAO+vehicle, and MCAO+A151 groups. Scale bars, 50 μm. (E) Bar graph shows the relative immunofluorescence intensity of GSDMD, caspase-1, and IL-1β in the brains of indicated groups. n = 6 mice per group. DAPI staining: nuclei.
Smad4 regulates diencephalic astrocyte proliferation and neurosphere formationA Reactome Pathway analysis for all 174 transcription factor genes with higher expression in DIE (upper) and all 205 genes higher in CTX GM (lower) astrocytes.Figure 9A Reactome Pathway analysis for all 174 transcription factor genes with higher expression in DIE (upper) and all 205 genes higher in CTX GM (lower) astrocytes. B t-SNE visualization of DIE astrocytes isolated by ACSA-2 MACS showing Smad4 expression (scaled log normalized read counts). C Example of a Smad4/GFP double-positive astrocyte (indicated by arrowhead) in a single optical section of DIE from 3 mo old GLASTCreERT2/eGFP mice 21 dpt immunostained as indicated on top of the panel. Orthogonal projections of the area highlighted in left panel of C shown at higher magnification in the right panel. Scale bar: 15 µm. D Example of a proliferating (PCNA+, arrowhead) astrocyte in a single optical section of DIE from 3 mo old GLASTCreERT2/eGFP mice 21 dpt immunostained as indicated on top with single channels shown in middle and right panels. Scale bars: 15 µm. E Histogram depicting percentage of PCNA positive cells of all GFP positive cells in the DIE of the genotype indicated on the x-axis (n = 6 animals per genotype; 5 ROI covering the diencephalon on a total of 3 slides were analyzed per animal; PCNA negative and positive cells comparing wt/wt vs. fl/fl animals, adjusted P-value = 0.002). Data information: data are presented as mean ± SEM. Each dot represents one n. * P < 0.05, ** P < 0.01 nonparametric 2way ANOVA followed by Sidak's multiple comparisons test F, G Histograms depicting percentage of the neurosphere-generating cells obtained from the DIE (F) or SEZ (G) of GLASTCreERT2/Smad4fl/fl/eGFP or GLASTCreERT2/Smad4WT/WT/eGFP, as indicated on the x-axis (n = 3-4 animals for SEZ, 4 to 8 technical replicates analyzed per animal; 4 animals for DIE, 8 technical replicates analyzed per animal). Data information: data are presented as mean ± SEM. F: GFP negative and GFP positive cells comparing wt/wt vs fl/fl animals, adjusted P-value = 0.0408; G: GFP negative and GFP positive cells comparing wt/wt vs fl/fl animals, ns). Each dot represents one n. * P < 0.05, ** P < 0.01 nonparametric 2way ANOVA followed by Sidak's multiple comparisons test H Histogram depicting the number of secondary neurospheres generated from 10000 Smad4f/f neurosphere cells at 7 days after infection with MLV-based retrovirus containing either CAG-NLS-Cre or only CAG-GFP (n = 11 cultures / animals, 3 technical replicates per condition analyzed; control vs. Cre, P-value = 0.0149). Note the decrease in DIE astrocyte proliferation in vivo and in neurosphere formation upon Smad4 deletion Data information: data are presented as mean ± SEM. Each dot represents one n. * P < 0.05, ** P < 0.01 nonparametric Mann-Whitney test).
Figure 3(A) Concentration of gut absorbed Evans Blue in serum 24 hours after oral administration in Regular-chow Diet (RD)- and high-fat diet (HFD)-fed mice, treated with vehicle or with antibiotic treatment; n= 6(RD), 5(HFD), 5(HFD+AB), 5(RD+AB); CI 99%. (B) Activation of PRRs induced by stimulation with serum isolated from mice fed RD or HFD for 7 weeks and receiving vehicle or neomycin for 3 weeks; fold RD; n=3 for all groups; CI 99%. (C) Serum cytokine profiles determined by Bio-Plex assay (Bio-Rad) of IL-1β, n= 11(RD), 12(HFD), 12(HFD+AB), 11(RD+AB); CI 95%; IL-6, n= 11(RD), 12(HFD), 12(HFD+AB), 11(RD+AB), CI 99.9%; TNF-α n=11 for all groups, CI 99%; IL-17A n= 11(RD), 12(HFD), 12(HFD+AB), 11(RD+AB); CI 95%; and IFN-γ; n= 12 for all groups; CI 95%. (D) mRNA expression of Il-1β; n= 5(RD), 7(HFD), 7(HFD+AB), 3(RD+AB); CI 95%, Il-6; n= 5(RD), 5(HFD), 4(HFD+AB), 4(RD+AB); CI 95%, Tnf-α; n= 6(RD), 9(HFD), 4(HFD+AB), 5(RD+AB); CI 95% and Vegf-a; n= 6(RD), 5(HFD), 5(HFD+AB), 6(RD+AB); CI 95% in choroids.
Spike-in of erythrocyte and platelet fractions into pure plasma.Figure 2Dilution and analysis scheme. Protein intensities were Z-scored across the dilution series for the 29 quality markers of the erythrocyte panel and (C) for the 29 markers of the platelet panel as a function of their spike-in proportion to plasma. Whiskers indicate 10-90 percentiles and horizontal lines denote the mean. (D) Correlation of erythrocyte count to the 'contamination index' for the erythrocyte marker panel. (E) Correlation of platelet count to contamination index for the platelet marker panel.
ECE2 KO and enzymatic inhibition cause a change in cell fate and neuronal positioning in COs.Figure 3Brightfield images of 60d old CTRL and ECE2 KO COs show normal formation of neuroepithelial structures. ISH for ECE2 confirms the absence of ECE2 mRNA in 60d old ECE2 KO COs (pink dotted line indicates position of the CP'). , FACS analysis of 60d old ECE2 KO COs reveals an increase in progenitors and decrease in neurons (n=7-8 samples of 3 pooled organoids each from 3 different batches; **P < 0.01; ***P < 0.001 in One Way ANOVA and Tukey Pairwise Multiple Comparison). Example images of IHC for PH3+ cells in 60d old CTRL and ECE2 KO COs. Quantification of PH3+ cycling cells in 60d old COs, normalised to length of the apical surface of ventricle-like structures, shows an increase upon ECE2 KO (Data shown as box plots with median as black line, mean as red line; n=number of analysed ventricles from 2 batches of COs; One Way ANOVA: **P = 0.004). In ECE2 KO COs, more ventricles are disorganised in terms of neuronal localisation as revealed from MAP2+ cell bodies in the progenitor zone (n=number of ventricles from 2 independent batches; two-tailed Mann Whitney U (U = 458) and chi-square test: χ2(2)=25.42, ****P < 0.0001).
Figure 5Agarose gel electrophoresis of LOR, FLG, and LCE1B promoter fragments amplified by end-point PCR after ACTL6A and BRM (left), or BRG1 (right) chromatin IP in proliferating keratinocytes. Agarose gel electrophoresis of the same PCR products described in (A) after ACTL6A (left), BRM (middle), and BRG1 (right) chromatin IP in uc.291 silenced and differentiated HEKn cells. One representative experiment of three is shown. Relative quantification by RT-qPCR of LOR, FLG, and LCE1B mRNA in uc.291 and ACTL6A silenced and differentiated HEKn cells. Data shown are the mean ± s.d.; n=3 (technical); *p<0.05, Student's t-test. Quantification by Real Time PCR of LOR, FLG, and LCE1B promoter fragments after H3K27ac chromatin IP in the same samples described in (B). Data shown are the mean ± s.d.; n=3 (technical); *p<0.05, Student's t-test. WB analysis of Loricrin expression in uc.291 and ACTL6A silenced and differentiated HEKn cells. β-actin was used as loading control. One representative experiment of three is shown.
Figure 3A-H. To examine the role of the p38-mediated innate immune signaling pathway in the long-lifespan of the nuo-6 and isp-1 mitochondrial mutants, we crossed the long-lived mitochondrial mutants to worms with mutations in nsy-1, sek-1, pmk-1 and atf-7(gof). In each case, mutation of genes involved in p38-mediated innate immunity signaling markedly reduces the lifespan of the long-lived mitochondrial mutant but have little or no impact on wild-type lifespan. In panel G, we utilized pmk-1 RNAi instead of the pmk-1 mutation because of the close proximity of isp-1 and pmk-1 on the same chromosome. EV indicates empty vector. The lifespan results for panels A, B, D, E, F, and H were performed in a single parallel experiment. Results are from a minimum of three biological replicates per strain. Data information: Statistical significance for survival plots was determined with the log-rank test. p-values indicate significance between red and purple lines. Control strains are shown in multiple panels for direct comparison.
Fig.Figure 2HEK 293 cells were transfected with a C-terminally VSV-tagged NrCAM construct or empty vector. Cells were then treated with DAPT (1 µM), GI254023x (5 µM), either substances or solvent for 24 h. The C-terminal NrCAM-fragment was detected with an antibody against the VSV-tag. Representative Western Blots are shown.
Figure 4(A) Primary cortical neurons were transduced with shTDP or control (DIV2+7) and subjected to quantitative proteomic analysis. Volcano plot shows up and down regulated proteins from three biological replicates. Significant changes are colored. Compare Table EV1. (B) Selected significantly enriched annotation between TDP-43 knockdown and control in proteomic data (complete data in Table EV1) (C-E) Primary hippocampal neurons (DIV6+4) were transduced with lentivirus expressing shRNA targeting TDP-43 or a control. Immunoblots with the indicated antibodies (C). Quantification of VPS4B and TDP-43 protein level using densitometry (n = 4). Expression was normalized to the housekeeping gene β-actin (D). (C-E) Primary hippocampal neurons (DIV6+4) were transduced with lentivirus expressing shRNA targeting TDP-43 or a control. VPS4B mRNA level were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping genes YWHAZ and PGK1 (E). (F) Primary hippocampal neurons (DIV6+3) were transfected with either shTDP or shCtrl and iRFP670. Immunostaining with the indicated antibodies. Scale bar represents 50 µm. (G) iPSC-derived human neurons were transduced with shRNA targeting human TDP-43 (shTDP) or control (shCtrl) seven days after thawing for three days. VPS4B levels were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping gene YWHAZ and PGK1 (n = 3).
Figure 4A Plants expressing the truncated asDOG1 promoter-deficient version pDOG1shDOG1::LUC are more resistant to drought than plants expressing the full-length genomic version pDOG1::LUC-DOG1. Three independent lines are shown for each construct, following 10 days of water deprivation. Scale bar, 2 cm. B RT-qPCR quantification of selected drought marker genes in mock- and ABA-treated Col-0 (WT) and two independent pDOG1shDOG1::LUC transgenic lines. Error bars represent standard deviation and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.
Figure 1Representative immunofluorescence (IF) staining of Ki67 and TDP-43 in the regions of frontal cortices from 6-mon-old WT and FTLD-TDP Tg mice. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; upper panel in blue) or neural marker NeuN (lower panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Quantification of cells or neurons with Ki67 immunoreactivity and TDP-43 mislocalization from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, ***p = 0.0007 by t-test. Representative data of reverse-transcription PCR (left panel) or Western blot (right panel) for cell cycle-related genes and semi-quantification of the expression levels in the frontal cortices and hippocampus from the 6-mon-old WT and FTLD-TDP Tg mice. N = 5 mice per group, data are presented as mean ± SEM (%), statistical analysis by multiple t-test with FDR correction, Q = 1%. *P < 0.05, **P ≤ 0.01; ***P ≤ 0.001 and ****P ≤ 0.0001
Figure 3(A) A comparison of the IFITM locus on chromosome 11 of the human reference genome (GRCh38.p5) and the corresponding locus in the Bushbaby reference genome (OtoGar3) visualized using Genomicus v84.01. Synteny is observed between human IFITM and Bushbaby IFITM, as evidenced by the conserved flanking genes B4GALNT4 and ATHL1. The Bushbaby locus lacks IFITM2 and IFITM1 genes. In addition, it contains two adjacent IFITM3 genes, as well as a third IFITM3 gene located in a different genomic context, indicative of gene duplication events. (B) A gene gain/loss tree summarizing the phylogenetic history of the IFITM gene family in primates, using annotated reference genomes in Ensembl. The number at a branch tip refers to the number of IFITM genes present in a given extant species, while the number at each node refers to the number present in an ancestral species. Differences in gene number at tips and nodes represent significant gene gain events (expansions) or gene loss events (contractions) based on a p-value calculation of <0.01 determined by CAFE (Computational Analysis of gene Family Evolution) (55). The makeup of the IFITM repertoire for each species, as determined by comparative genomics and the IFITM gene family tree in Ensembl, is detailed to the right. When multiple copies of IFITM genes were identified, the total number is listed inside the box. The gene numbers estimated for the Mouse Lemur and Bushbaby in Ensembl were adjusted following phylogenetic analysis of IFITM homologs in Genomicus as well as BLAST searches of the NCBI GenBank database. Blue and red arrows indicate the emergence of IFITM1 and IFITM2 genes, respectively.
a,b) Transmission electron microscopy of morphological changes in mitochondria (arrows) in Map1lc3b−/− BMDMs (a) or Becn1+/− BMDMs (b) left untreated or incubated for 6 h with LPS (10 ng/ml) and then stimulated for 30 min with ATP (1 mM).figf2(a,b) Transmission electron microscopy of morphological changes in mitochondria (arrows) in Map1lc3b−/− BMDMs (a) or Becn1+/− BMDMs (b) left untreated or incubated for 6 h with LPS (10 ng/ml) and then stimulated for 30 min with ATP (1 mM). Outlined areas (middle) enlarged at right. Scale bars, 500 nm. (c) Flow cytometry of Map1lc3b−/− and Becn1+/− macrophages left unstained or labeled with MitoSOX. (d) Flow cytometry of macrophages left untreated or stimulated with LPS and ATP; cells were stained for 15 min with MitoTracker Deep Red and MitoTracker Green before ATP treatment. Data are representative of three experiments.
Phosphorylation of STARD3 in its Phospho-FFAT motif governs membrane attachment and cholesterol transfer in in vitro reconstituted assays.Figure 5A: Description of the experimental strategy. LA liposomes (endosome-like) are decorated with cSTD3 or pS209 cSTD3 owing to covalent links with MPB-PE (1.2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl) butyramide]), and mixed with LB liposomes (ER-like) covered by VAPHIS6 attached to DOGS-NTA-Ni2+ (1.2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl], nickel salt). For dehydroergosterol (DHE) transport experiment, LB liposomes also contain DHE and a dansyl phosphatidylethanolamine (DNS-PE). The transport of DHE from LB to LA liposomes is followed by measuring the change in FRET from DHE to DNS-PE. B: SDS-PAGE gel and Western blot analysis of purified cSTD3 and pS209 cSTD3 proteins. Top: the gel was stained with Sypro Orange to visualize proteins and molecular weight markers. Bottom: two similar gels were blotted onto nitrocellulose and analysed for the presence of total and STARD3-pS209 using specific antibodies. C: Aggregation assays. LA liposomes (50 µM total lipids) were incubated for 5 minutes with cSTD3 or pS209 cSTD3 (380 nM). Then, LB liposomes (50 µM total lipids) and VAP-AHIS6 or VAP-A KD/MDHIS6 (700 nM) were successively added. Left panels: mean radius (black dots) and polydispersity (shaded area) over time. Right panels: size distribution before (grey bars) and after the reaction (black bars). D: DHE transport assay. DOPC (1.2-dioleoyl-sn-glycero-3-phosphocholine) liposomes (62.5 µM total lipids, LA) containing 3 mol% MPB-PE were mixed with cSTD3 or pS209 cSTD3 (475 nM). After 5 minutes, liposomes (DOPC/DOGS-NTA-Ni2+/DNS-PE/DHE liposomes 77.5/10/2.5/10 mol/mol, 62.5 µM total lipids, LB), covered or not with 1 µM of VAPHIS6 or VAP-A KD/MDHIS6, were added. FRET between DHE and DNS-PE in the LB liposomes diminishes as DHE is transported towards LA liposomes. The signal was converted into amount of DHE present in LB liposomes (in mol%). E: Initial DHE transport rate measured with cSTD3 or pS209 cSTD3 in the presence or absence of VAP-A HIS6 or VAP-A KD/MDHIS6. Data are represented as mean ± S.E.M. (n = 3 for cSTD3-VAP and n=4 for all other data). Mann-Whitney test (*P < 0.05).
TRF2 changes glycocalyx structure, which triggers MDSC activation A Analysis of Heparan Sulfate (HS) expression by FACS using the 10E4 antibody.Figure 6A Analysis of Heparan Sulfate (HS) expression by FACS using the 10E4 antibody. Data information histograms representative (left panels) and MFIs from three independent experiments (*p < 0.05 and **p < 0.01; Student's t-test) are shown +/- SEM. B Atomic Force Microscopic (AFM) analysis of glycocalyx and cell stiffness and glycocalyx length. Data information: In (B), Box plots Min to Max showing all points with median represent four independent experiments, each performed using at least 20 cells (*p< 0.05; Mann-Whitney test). C AFM images showing the Modulus heatmap (stiffness) in GFP-expressing or TRF2-overexpressing cancer cells. At each of the 128x128 force curve points, corresponding to an area of 10 μm2, the AFM software automatically displays the Sneddon modulus analysis while scanning the sample. The color bar on the left of the images shows a qualitative representation of the elastic map. D Analysis of pSTAT3 levels in MDSCs after co-culture with GFP-expressing or TRF2-overexpressing cancer cells treated with or without sodium chlorate (NaClO3). Data information: In (D), data represents mean of n = 3 independent experiments +/- SEM (***p < 0.001; Student's t-test).
Fig.Figure 3(A) ADAM10 fl/fl neurons were treated with an iCre (to induce an ADAM10 KO), or a control lentivirus at DIV2. At DIV7, cell surface proteins were labeled with biotin and enriched by streptavidin pull-down. The biotinylated proteins were detected by immunoblotting. Total lysates were analyzed to compare mNrCAM's surface/lysate levels. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (**** p < 0.0001, n = 7). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. (B) Primary murine neurons were treated with GI254023x (5 µM) or solvent, for 48h. The cell surface proteins were enriched like in A. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (**** p < 0.0001, n = 11). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown. (C) Workflow of the neurite outgrowth assay. (D) Representative pictures of single neurites at time-point 0h (DIV3) and 24h (DIV4). Neurons were infected 4h after plating with lentiviruses encoding GFP (to visualize the neurons) together with shRNA expression cassettes (sh1 and sh2) targeting either NrCAM or carrying a scrambled (scr) control construct. Images of neurites were taken at three days in vitro (DIV3) and 24h later at DIV4. In order to study the effect of ADAM10 on neurite outgrowth, neurons were treated with the ADAM10 inhibitor GI254023x, or vehicle (control), at DIV3, after taking the first pictures with an epifluorescent microscope. The differences in neurite length were calculated as absolute values (neurite length at 24h minus neurite length of 0h) for individual neurites passing through the middle channels of the chambers. Only neurites that had already entered the main channel at 0h and had not yet left those channels at 0h were considered. The red arrows indicate the start and the end of the respective length measurements. The scale bar indicates 40 µm. (E) One-way ANOVA with post hoc Dunnett's test. Given are mean +/- the standard error of them mean (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, n-numbers for each condition are shown in the graph in 3E).
Increased NLRP3, complement C1q and C5b-9 in CNV lesions of VEGF-Ahyper mice.Figure 7A. NLRP3 is expressed in RPE cells at sites of CNV lesions (arrow; white).B. Lack of NLRP3 staining in VEGF-Ahyper/NLRP3-/- mice (control). Scale bars 100 μm. C. Expression of NLRP3 [*p-value: 0.0478] and IL-1β [*p-value: 0.0398], but not of IL-18 [p-value: 0.3529] is increased in RPE/choroid lysates from VEGF-Ahyper mice when compared to matched control littermate mice. N=7 mice/group (3 independent experiments). Graphs show mean ± SEM. D.-G. Complement pathway activation (immunolabeling for the product of complement pathway activation C5b-9, an inducer of the NLRP3 inflammasome) is observed in CNV lesions of VEGF-Ahypermice (white arrow in E.) but not in age-matched control mice (D.) Co-localization of C5b-9 in the same CNV lesion is observed with complement C1qA (the initiator of the classical complement pathway and an inducer of the NLRP3 inflammasome) (F.), and with TLR2 (G.) (white arrows). Autofluorescence in red in D. and E. CD31+vessels in red in I. and J. Yellow arrows show site of choroid. Scale bars 50 μm.
Figure 7(A) RPE1 cells were transfected as indicated, followed by incubation in serum-starvation media in the presence or absence of cholesterol/MCD for 24 h, and then immunostained for Rab8 (red) and γ-tubulin (green). Scale bar, 10 μm. Representative magnified images are shown from cells labeled with white asterisks. (B) Quantification of the percentage of cells with Rab8 localized to the centriole Data represent mean ± SD (n=3 experiments), 200 cells were scored per condition per experiment, *P < 0.05, Student's t-test. (C) (Upper panel) Cells were transfected and cell lysate was incubated with purified GST-JCF1 (RBD) fusion protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by western blot with Rab8 antibody. (Lower panel) The intensity of bands was quantified by Image J software. The amount of GTP-Rab8 were normalized to the control level. The bar graph represents mean ± SD (n=3 experiments). *P < 0.05, Student's t-test. (D) Cells were transfected and immunostained for IFT20 (red) and γ-tubulin (green). Scale bar, 10 μm. (E) Quantification of IFT20 intensity at the centriole Data represent mean ± SD (n=3 experiments), 150 cells were scored per condition per experiment, *P < 0.05, Student's t-test. (F) A proposed model showing the importance of cholesterol for primary ciliogenesis.