EMBO/sd-character-level-ner huggingface dataset:
Figure 1(A) Representative image of a typical young WT cortico-hippocampal slice (350 µm) from co-culture (14 DIV) stained with DAPI (gray) to visualize nuclei (left). Dentate gyrus (DG), cornus ammonis 1 (CA1) and cortical (CTX) regions are indicated. An example of the co-culture dish is shown on the right. Co-culture includes 2 young WT (*, young co-culture) and 2 old APPPS1 (+, old co-culture) brain slices. (B) Immunofluorescence analysis of young WT and old APPPS1 slices cultured alone (young alone, old alone) or in co-culture (young co-culture, old co-culture) at 7 days in vitro (DIV) stained with propidium iodide (PI, red) reveals reduced cell viability in old brain slices compared to young.(C) Quantitative analysis of PI positive cells in young WT and old APPPS1 slices cultured alone (white and black bars, respectively) or in co-culture (grey and white squared bars, respectively) at 7 and 14 DIV. The values are expressed as percentages of PI positive cells from the total number of DAPI positive cells. The values represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (n.s.= not significant; ***P < 0.001). (D-F) Immunofluorescence analysis of young WT and old APPPS1 slices cultured alone or in co-culture at 7 DIV and immunostained using neuronal (NeuN), astrocytic (GFAP) and microglial (CD68) markers (green) reveals reduced NeuN and GFAP and increased CD68 immunosignal. Scale bar: 75 µm
Figure 4A. Mean ± SEM cholesterol level in plasma membrane fractions of hippocampal slice cultures from wt and NPC1nmf164 mice with or without cLTP induction (n=6, unpaired Student t test, *pwt = 0.0402 B. Western blot of NPC1 and ACTB in extracts of cultured hippocampal neurons from wt mice transfected or not with sh-scr or sh-NPC1 RNAs. Black bars indicate that the sh-NPC1 lane was not consecutive to the others but belongs to the same Western blot. Graph shows the 2 biological replicates mean ± SEM of NPC1 level normalized to ACTB as a percentage of the wt, non-transfected controls. C. Representative images of the cholesterol binding probe mCherry-D4 before and after cLTP in cultured hippocampal neurons from wt mice transfected with sh-scr or sh-NPC1 RNAs. Z-stack images acquired using a Nikon A1R+ confocal microscope were used to generate 3D images of the neurons. Western blots against NPC1 actin-β (ACTB) in total extracts (T) and biotin-streptavidin immunoprecipitates (S) from synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not. Graphs show mean ± SEM of the levels of biotinylated-surface NPC1 as a percentage of the total amount of each protein (n=3 mice, 3 month-old, 2-way ANOVA, *pNPC1 = 0.0251 Western blots against CYP46A1 and actin-β (ACTB) in total extracts (T) and biotin-streptavidin immunoprecipitates (S) from synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not. Graphs show mean ± SEM of the levels of biotinylated-surface CYP46A1 as a percentage of the total amount of each protein (n=3 mice, 3 month-old, 2-way ANOVA , *pCYP46A1 = 0.0371). F. Representative images of GluA1 immunocytochemical staining before and after cLTP in non-permeabilized (surface) and permeabilized (total) cultured hippocampal neurons expressing sh-scr or sh-NPC1 RNAs. Graph shows mean ± SEM GluA1 surface staining with respect to total in cLTP conditions, as a percentage of the sh-scr baseline (n=20 images per condition from 2 different experiments, 2-way ANOVA, ***psh-scr < 0.001).
Figure 5(A) Larval NSCs of control (ctrl; yw) and ebd1240 at 24h ALH were stained for Deadpan (Dpn) and Miranda (Mira). (B) Quantifications of cellular extensions for genotypes in (A). Control: n=886, 11 BL; ebd1240: n=864, 12 BL. (C) Larval brains of control (ctrl; yw) and ebd1240 at 24h ALH were labelled with Dpn and EdU. (D) Quantifications of NSC without EdU incorporation for genotypes in (C). Control: n=1034, 12 BL; ebd1240: n=1139, 16 BL. Data information: Data are presented in (B, D as mean ± SD. n numbers in (B, D, were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. Statistical analyses were done comparing two different genotypes using Mann-Whitney test (B, D ns: non-significant; *P < 0.05; ***P < 0.001. (E) Larval NSCs of grainyhead (grh)>CD8-GFP at 0h, 24h and 48h ALH were stained for CD8, Dpn, and Ebd1. Yellow dotted boxes indicate the region of zoomed-in images. Data information: Scale bars, 15 μm (E), Quantifications of nuclear/cytoplasmic Ebd1 (F) In (F), n=1044, 14 BL at 0h ALH; n=712, 10 BL at 24h ALH and n=1122, 11 BL. Data information: Data are presented in F, as mean ± SD. n numbers in F, were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. Statistical analyses were done comparing two different genotypes using Mann-Whitney test F). statistical analyses were done comparing the percentage at later time points with the ones at 0h ALH. ns: non-significant; *P < 0.05; ***P < 0.001. G) Quantifications of the corrected total cell fluorescence (CCF) of Ebd1 in NSCs (G) In (G), CCF = Integrated Density - (area of selected cell × mean fluorescence of background readings). For CCFs of Ebd1, 128.5±140.7 (n=162) at 0h ALH, 938.7±1176.8 (n=150) at 24h ALH and 1868.2±1025.1 (n=161) at 48h ALH. Data information: Data are presented in G, as mean ± SD. Statistical analyses were done comparing among different genotypes using ordinary one-way ANOVA (G, ns: non-significant; *P < 0.05; ***P < 0.001. (H) Larval brains of control (ctrl; yw), ebd1-Flag overexpression under insc­-Gal4, pr-set7716/Df(3R) ED5644 (716/Df) and pr-set7716/Df with ebd1 induced with insc-Gal4 at 24h ALH were labelled with Dpn and EdU. (I) Quantifications of NSCs without EdU incorporation for larval brains in (H). Control: n=1162, 13 BL; insc>ebd1: n=997, 11 BL; pr-set7716/Df: n=928, 13 BL and pr-set7716/Df with ebd1 induced with insc-Gal4: n=1284, 16 BL. Data information: Data are presented in I) as mean ± SD. n numbers in I) were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. Statistical analyses were done comparing among different genotypes using ordinary one-way ANOVA I). ns: non-significant; *P < 0.05; ***P < 0.001.
WT and NRBF2 KO MEFs were treated with DMSO, 1 μM thapsigargin (TP) or 1 μM tunicamycin (TN) for 24 or 48 h.f7WT and NRBF2 KO MEFs were treated with DMSO, 1 μM thapsigargin(TP) or 1 μM tunicamycin (TN) for 24 or 48 h. (a) Then cells were either imaged under phase-contrast microscope or (b) stained with propidium iodide (PI) and subjected to cell death rate analysis by flow cytometry. The representative images of cell morphology after treatments and quantitative analysis of PI-positive cells rate show marked increase of cell death in the NRBF2 KO MEFs compared with WT MEFs. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=5). Scale bars, 100 μm; Con, control. (c) Re-introducing NRBF2 to NRBF2 KO MEFs reverses the vulnerability of cells to ER stress. NRBF2-CFP stably expressing cells were established in the NRBF2 KO background. Cells were treated with DMSO or 1 μM TP for 24 h then stained with PI and subjected to cell death rate analysis by flow cytometry. The cell death rate in CFP-positive (+) population (NRBF2-CFP expression) and negative (−) population (no NRBF2-CFP expression) were recorded and analysed, respectively. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=3). (d) ER stress inducers trigger caspase-8 and caspase-3 activation in NRBF2 KO MEFs. MEFs were treated with DMSO, 1 μM TP or 1 μM TN for 24 h, and then immunoprobed with indicated antibodies. Marked accumulation of cleaved caspase-8 (C-Cas 8; p43 and p18 units) and cleaved caspase-3 (C-Cas 3) are observed in the NRBF2 KO MEFs but not WT MEFs treated with TP or TN. This is a representative image from three independent experiments. (e) C-Cas 8 preferentially localizes at p62 aggregation foci. MEFs were treated with DMSO or 1 μM TP for 24 h, and then co-stained with an anti-p62 antibody and an anti-cleaved caspase-8 antibody. The C-Cas 8 fluorescence signals are triggered in NRBF2 KO MEFs treated with TP and preferentially localize at p62 aggregation foci. Scale bars, 20 μm. This is a representative image from three independent experiments.
Figure 8A. In the environment with low concentrations of TGF-β1, activation of TGFβ receptor induces miR-21, which blocks the translation of Smad7, thus relieves the suppression of TGF-β1 expression by Smad7, and promotes TGF-β1 release from MSCs. B. Overactivation of TGFβ receptor in MSCs by high environmental TGF-β1 down-regulates miR-21 and thus no longer inhibits Smad7 translation and leads to close the TGF-β1 signal circuit. Such adaptive response is disturbed by deliberately changing the expression of components in the signaling pathway, such as (1) silencing TGF-β1 expression in MSCs by TGF-β1 siRNA, (2) blocking TGFβ receptor activity by chemical inhibitor SB431542, (3) silencing Smad7 expression by Smad7 siRNA, (4) over-expression of miR-21 by miR-21 mimic, and (5) knock-down miR-21 by miR-21 inhibitor.
VcdR is a direct inhibitor of the synthesis of PTS sugar transport proteinsA-D.Figure 4A-D. Relative fluorescence intensities (y-axis) of E. coli strains harboring the gene-specific translational reporters for ptsG (A), treB (B), nagE (C) and ptsHI (D), fused to sfGFP, combined with either an empty vector control (pCtrl) or vcdRP expression plasmids (pVcdRP, pVcdR, pVcdP and pVcdRP Δ4C, x-axis). Cells were grown in LB to OD600 of 1.0 and fluorophore production was measured. The fluorescence of pCtrl (for each reporter fusion) was set to 1. E-H. Predicted base-pairing sequences for ptsG (E), treB (F), nagE (G) and ptsHI (H) to VcdRP. The numbers indicate the distances relative to the TSS for VcdRP, and the start codons of the respective mRNA sequences. Underlined is the conserved stretch of four consecutive cytosines that corresponds to the seed region indicated in (1C). The M2* and M2 mutations refer to the site-directed mutagenesis of two guanines/cytosines in positions 2 and 3 of the seed region to two cytosines/guanines in the corresponding mRNA/VcdRP, respectively. I-L. Relative fluorescence intensities (y-axis) of E. coli strains harboring the gene-specific translational reporters as in (A-D) or the corresponding M2* variant for each gene, combined with an empty control plasmid or vcdRP expression plasmids (pVcdRP and pVcdRP M2) measured using plate reader (I-K) or by Western blot analysis (L). The fluorescence of pCtrl (for each reporter fusion) was set to 1.
Blood vessel abnormalities arise during progressive glioma growth.Figure 1A. Glucose transporter1 (Glut1) immunohistochemistry on sections of 5 weeks growth glioma in ROSAmT/mG::Pdgfb-iCre mouse (50 µm depth stack). Hypoxic tumor cells upregulate Glut1. B. Still image of two-photon live imaging on 2 weeks growth glioma implanted in ROSAmT/mG::Pdgfb-iCre mouse demonstrating tip cell filopodia extension indicative of sprouting. See Supplementary movie 1. C. Representative images of two-photon live imaging of the same glioma area of the same mouse on 2 and 5 weeks growth glioma (BFP positive) implanted in ROSAmT/mG::Pdgfb-iCre mouse (350µm depth stack). Note differences in network complexity and vessel diameter. D. Blood vessel diameter quantification: sprouting blood vessels at 2 weeks growth present caliber comparable to vessels in the healthy brain (hb). At 5 weeks growth, tortuous blood vessels in the tumor are significantly more dilated than in the healthy brain (n=8 mice). E. Branchpoint quantification during glioma growth (n=8 mice). F. Two-photon live imaging of vessel perfusion using FITC-dextran IV injection in ROSAmT/mG mice (300µm depth stack). G. Quantification of unperfused vessel segments (asterisk) shows increase during glioma growth (n=5 mice). H. Tumor bearing brain overview images after Miles assay. I. Late stage glioma growthvessels are leaky compared to early tumor growth (n=5 mice per group). Statistical analysis: D.E.G.I. one-way ANOVA followed by multiple comparisons Tukey's test. Error bars: meanSD. Scale bars: A: 250µm; B: 50µm; C-F: 150µm; G: 0,5cm.
Figure 3A Mitochondrial translation products were labeled using a FUNCAT approach and [35S]Met labeling in control and TSFM patient fibroblasts. Representative epifluorescent microscopy and the autoradiography images are shown. Scale bars, 50 µm. Changes in mitochondrial protein synthesis were quantified for both approaches. For microscopy image analysis, >300 cells from N = 3 independent experiments were analyzed for each cell type. Mann-Whitney U test, ****p < 0.0001. For radiolabeling analyses, N =3 independent samples were quantified for each cell type. Student t test, **p < 0.01. Error bars, +/- SEM. B As in A using control and COA6 KO HEK cells. Scale bars, 25 µm. For microscopy image analysis, ****p < 0.0001, and for radiolabeling experiments, *p < 0.05 were calculated. Error bars, +/- SEM. C Mitochondrial protein synthesis was monitored in hippocampal neurons, using ATTO 590-azide (magenta) labelling and cells immunostained for TOM20 (green). A representative deconvolved STED microscopy image is shown. Scale bars, 1 µm.
a) Relative acetylation (normalized to controls at each day) of indicated histone H3 Lys residues determined by quantification of immunoblot analysis.figf4(a) Relative acetylation (normalized to controls at each day) of indicated histone H3 Lys residues determined by quantification of immunoblot analysis. A representative blot using site specific antibodies is shown in b. Data represent means ± s.e.m. (n = 3). Serial dilutions of protein extracts were applied in western blots to verify linearity before quantification (examples are shown in Supplementary Information, Fig. S4). For calculation details see section on Methods. P values indicate the result of a two-factor ANOVA corrected by Bonferroni post hoc test. (b) Immunoblot of whole-cell extracts of wild-type cells chronologically aged to designated time-points with (+) or without (−) spermidine application. Blots were probed with antibodies against total histone H3 or H3 acetylation sites at the indicated Lys residues. Full scans of blots are available in Supplementary Information, Fig. S5. (c) Relative acetylation (normalized to wild-type controls at each day) of indicated histone H3 Lys residues of Δspe1 cells (open bars), compared with wild-type cells (closed bars) during chronological ageing. Data represent means ± s.e.m. (n = 3). P values indicate the result of a two-factor ANOVA corrected by Bonferroni post hoc test. (d) Relative acetylation (normalized to day 1) of histone H3 Lys 18 residue of chronologically ageing wild-type cells. Data represent means ± s.e.m. (n = 3). (e) Relative acetylation (normalized to controls) of histone H3 Lys 14 and 18 residues of cultured human PBMC after 6 days of incubation with or without 20 nM spermidine. Data represent quantification of two independent experiments performed with cells obtained from two different donors. (f) Immunoblot analysis of liver cell extracts (applied in serial dilutions) obtained from mice fed with 3 mM spermidine for 200 days and respective control mice of the same age. Blots were probed with antibodies recognizing total histone H3 (C-terminal epitope) or specific for acetylated lysine 18 residue (N-terminal epitope). Full scans of blots are available in Supplementary Information, Fig. S5.
Figure 2  A. Enrichment and depletion of different amino acids in the random library (log2 of frequencies of functional over non-functional sequences)    B   In the boxplots, the central line shows the median. Statistical significance was assessed using Wilcoxon test. Boxplots of the distribution of the values of: B. length C. pI D. hydrophobicity E. disorder content and F. helicity for sequences that are functional (green) and non-functional (red  .-G Statistical significance was assessed using Wilcoxon test  G. Enrichment of 9aa TAD motif in functional vs. non-function sequences; ratio of with to without 9 aa TAD in functional to non-functional sequences ((219/520)/(13384/50001)
Experimental design for quantitative proteome-wide solubility measurements after heat shock.Figure 1A Dynamic SILAC, heat treatment and recovery. K562 human cells were grown at 37°C in light SILAC medium. 90 minutes before heat treatment the medium was changed to heavy SILAC containing stable heavy carbon and nitrogen isotopes in arginine and lysine amino acids. These heavy amino acids are incorporated into newly synthesized proteins while the light versions remain in pre-existing proteins. A control sample was collected (pre-shock) prior to partitioning cells for heat treatment. The cells were treated either with heat shock (44°C) or mock shock (37°C) for ten minutes. After heat treatment, cells were allowed to recover at 37°C and samples were collected at different time points. B Sample processing. Samples were lysed with mild detergent (0.8% NP-40), soluble proteins were extracted and digested to tryptic peptides. Peptides were labelled with TMT labels and pooled. After offline reversed phase fractionation, samples were analyzed with mass spectrometer. C Quantification of soluble protein fraction. MS1 scan allows to separate peptides from newly synthesized proteins (heavy) and pre-existing proteins (light). MS2 scan allows for peptide (and later protein) identification and, based on TMT reporter ion intensities, quantification from different samples. For MS2 scan, a hypothetical example is shown for aggregating and disaggregating protein from pre-existing (light) fraction.
Figure 4A. Raw 264.7 macrophage cells were transfected with Flag(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain(PI(3,4,5) P3 marker), or eGFP(C-term)-SARA-Fyve domain((PI3P marker), or eGFP(C-term)-PLC-PH domain (PI(4,5) P2 marker, epifluorescence, shown in blue) followed by anti-Flag immunofluorescence (shown in green) and pT73-Rab10 antibody (shown in red). To evaluate colocalization of phospho-Rab10 with early endosomes or late endosome/lysosome markers, immunofluorescence detection was performed for endogenous EEA1 or Lamp1 (shown in blue). Raw 264.7 macrophage cells were further fed 0.05 mg mL-1 of 70 kDa TRITC-conjugated dextran (epifluorescence, shown in blue) or 0.05 mg mL-1 BODIPY conjugated ovalbumin for 30min prior to immunofluorescence analysis. Representative images were selected from 25 cells analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are "High Mag" panels that show individual vesicles analyzed. Scale bars represent 10 μm and 1 μm for "High Mag". Positive colocalization signal (depicted as white) in the "CoLoc (pRab10/Rab10)' and 'CoLoc (pRab10/Marker)' columns are indicated. White arrow heads highlight vesicles scored as co-positive between pT73-Rab10 and Rab10 or the indicated marker. B. 25 cells across three independent experiments were analyzed for each condition, with each dot representing the mean of the vesicle analysis across individual cells. The group means of %pT73-Rab10 vesicles positive for the indicated marker are given, with error bars showing ± SEM.
Meth Induces Autophagosome Accumulation in Dendritic Cells.Figure 3(A) Accumulation of GFP-LC3 labeled autophagosomes in Meth- or Clq-treated dendritic cells at 2 h and 24 h. Fisher's exact test P values versus control (n =300-400 cells, mean ± SEM, *p < 0.05, ***p < 0.0001). (B) GFP-LC3 labeled autophagosome formation in different concentrations of Meth after 24 h treatment. Similar results were obtained in three independent experiments. Fisher's exact test versus control (n = 300-400 cells, mean ± SEM, ***p < 0.0001). (C) Representative fluorescence micrographs of GFP-LC3 decorated autophagosomes in cells treated with 50 μM Meth or 10 μM Clq for 24 h. Arrow denotes examples of autophagosomes. Scale bar, 10 μm.
VcdRP regulates central metabolism genesA.Figure 3A. A schematic representation of the three VcdRP and VcdR/P variants used for the transcriptome analysis. Left to right: first, the native VcdRP variant which harbors both the small protein as well as the base-pairing region. Next, a STOP codon was introduced in the 3rd codon of vcdP ORF as a readout for VcdR devoid of VcdP. Finally, the sequence of the VcdP codons was scrambled such that the correct small protein is translated but the nucleotide sequence of the scrambled vcdP variant is significantly altered when compared to native vcdRP. This variant also carries an artificial 5'UTR and a terminator. The arrows indicate the TSS. B. Western blot analysis (top) of CTX levels detected in secreted protein fractions of V. cholerae ΔhapR cells grown under AKI conditions and carrying the indicated plasmids. Coomassie-stained SDS gel (bottom) confirmed equal loading of the protein fractions. The normalized band intensities are indicated for each expression plasmid relative to pCtrl, which was set to 100%. C. A Venn diagram of the differentially expressed genes (DEG) among VcdRP (red), VcdR (yellow) and VcdP (blue). RNA samples were harvested from V. cholerae ΔvcdRP strains carrying pBAD-based plasmids depicted in (A), including an empty vector control. Genes with a total count cutoff >10 in all samples, with an absolute fold-change ≥ 2.0 and a FDR adjusted p-value ≤ 0.05 were considered to be differentially expressed. D. Gene enrichment analysis of the DEG shown in (C) using gene ontology analysis.
Figure 4A For macrophage depletion models, mice (six weeks) were injected with GdCl3 (15 mg/kg). After the induction of macrophage depletion in the mice, pancreatitis was induced by cerulein, LPS, and ANGPTL4 for 24 h (n = 10, each group). H&E staining of pancreatic tissue, and levels of amylase and lipase in the serum. Scale bar represents 100 µm B Immunohistochemistry for the macrophage marker CD11b and ANGPTL4 in pancreas tissues. Levels of ANGPTL4 in the serum were measured in the AP, SAP, and ANGPTL4 (4 mg/kg) groups of macrophage WT or depletion mice h (n = 10, each group). Scale bar represents 100 µm C Immunofluorescent staining of the F4/80 macrophage marker and quantification of macrophage-positive cells in the AP and SAP groups from the ANGPTL4 -/-and WT mice (n = 10). Scale bar represents 150 µm