EMBO/sd-character-level-ner huggingface dataset:
Figure 2(A) Synaptic terminal endocytosis was analyzed in WT and MPS-IIIA DIV14 hippocampal neurons by quantification of incorporated FM1-43 dye fluorescence in ~300 individual boutons (taken from 4-5 coverslips for each group). Fluorescence intensities were expressed as arbitrary units (A.F.U.) and displayed both as a distribution and as mean values ± s.e.m. (B) WT and MPS-IIIA hippocampal neurons (DIV14) were transfected with v-Glut1-pHluorin-mCherry plasmid and synaptic recycling was evaluated by the fluorescence change of the probe from 80-100 individual boutons (taken from 4-5 coverslips for each group). Fluorescence intensity was quantified at each bouton during KCl perfusion, normalized to the fluorescence value obtained upon rapid alkalization (NH4Cl perfusion) and expressed as ∆fluorescence (normalized to baseline; ΔFKCl/ΔF NH4Cl). Values were displayed both as fluorescence traces (means ± s.e.m. at each time frame) and maximum fluorescence after KCl perfusion (means ± s.e.m.). Representative panels on the right show the fluorescence intensity change in control neurons (WT). Pseudo-colour was applied to better reveal fluorescence changes.Note that NH4Cl alkalinizes all vesicles revealing the total (recycling + resting) pool in neuronal cells analyzed. (C) WT hippocampal neurons at DIV10 were treated with a lysosome inhibitor's cocktail for 3 days. The size of lysosomal compartment was evaluated in both treated and control untreated WT neurons by EM examination. Quantitation analysis was performed on 30 different images. (D, E) Endocytosis (D) in presynaptic boutons from treated and control untreated WT hippocampal neurons were monitored as in (A, B). (D, E) exocytosis (E) in presynaptic boutons from treated and control untreated WT hippocampal neurons were monitored as in (A, B). (F) EM analysis of synaptic terminals was performed in treated and control untreated WT neurons. The number of synaptic vesicles per synapse was quantified from ~20 different images (taken from 3 ultrathin sections for each group), normalized by the length of synaptic cleft and expressed as % of WT. Arrows indicate the synaptic cleft.
Figure 2A. Serum testosterone levels in the adult male and CtM Hanwoo (n = 10 in each group). The scattered dots in the bar graph represent the individual replicates. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the rumen, ileum, and colon, shown for the different segments of the gastrointestinal tract (B) C. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data or the rumen, ileum, and colon, combined data (C). D. Microbial dissimilarity (calculated based on the weighted UniFrac distance matrix, 100 values in each group) between the male and CtM groups in the different intestinal compartments. The lines, boxes, and whiskers in the box plot diagrams represent the median, first and third quartiles, and min-to-max distribution of replicate values, respectively. E. The relative abundances of abundant bacterial taxa (> 0.5% of the mean abundance).
Figure 6(A) Bioenergetic profile of human fibroblast cell lines. OCR (pmol/min/norm.unit) for cWT, cWT+ATAD1, PEX3-/-, PEX3-/-+ATAD1, cWT-ATAD1-/-, and PEX3-/- atad1-/- cells plotted against time (XF96e Extracellular Flux Analyzer, Mito-Stress-Test). 1 μM Oligomycin A, 0.15 μM FCCP, and 1 μM rotenone + 1 μM antimycin A (final concentrations) were sequentially delivered to the XF96e assay medium through injection ports in the sensor cartridge. The graph is the compilation of two independent assays, error bars show mean +/- s.d. (n=4, biological replicates). (B) Bar graph representation of basal respiration measured in (B) for cWT, cWT+ATAD1 , PEX3-/-, PEX3-/-+ATAD1, cWT-ATAD1 , and PEX3-/--atad1 cells. (C) Bar graph representation of maximal respiration measured in (B) for cWT, cWT+ATAD1 , PEX3-/-, PEX3-/-+ATAD1, cWT-ATAD1 and PEX3-/- -atad1 cells. (D) Bar graph representation of calculated respiratory spare capacity (uncoupled respiration minus basal respiration) measured in (B) for cWT, cWT+ATAD1 , PEX3-/-, PEX3-/-+ATAD1, cWT-ATAD1, and PEX3-/--atad1 cells. (E) Scatter plot representation of cardiolipin lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 11 detected cardiolipin species, log10 pareto-scaled is visualized. The horizontal lines represent the mean peak intesity of all 11 caridolipin species in each cell line. ­­(F) Scatter plot representation of a subpopulation of the phosphoethanolamine (PE) lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 20 phosphoethanolamine (PE) species (decreased in PEX3-/-), log10 pareto-scaled, is visualized. The horizontal lines represent the mean peak intesity of all 20 PE species in each cell line. (G) Scatter plot representation of ether-phospholipid lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 21 detected ether-phospholipid (plasmalogen) species, log10 pareto-scaled, is visualized. The horizontal lines represent the mean peak intesity of all 21 plasmalogen species in each cell line.
Figure 1A. Genetic interaction between condensin and Gcn5. Fivefold serial dilutions of fission yeaststrains were spotted onto complete medium. B Scheme of Gcn5-47. KAT: Lysine Acetyl-Transferase. Bromo: Bromodomain. C. Condensation assay. 3D distances between two fluorescently-marked loci on chromosome I were measured by live cell microscopy in fission yeast cells progressing from late G2 through mitosis. Time lapse recording for n>32 cells for one (wild type) or two (gcn5-47 and cut14-208) biological replicates were aligned to anaphase onset (t = 0) and average distances (± s.d.) plotted. Values are listed in Appendix Table S1. D. Chromosome segregation in gcn5-47 or cut3-477 mutant cells. Cells growing at 32°C were fixed and processed for immunofluorescence microscopy against Mis6-HA and Cdc11-GFP to reveal centromeres (Cen) and Spindle Pole Bodies (SPB), respectively. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI). Left panel: single mutant cells cut3-477 or gcn5-47 exhibiting chromatin bridges (b) or trailing chromatin (t). Bar: 5 µm. Right panel: frequencies of chromatin bridges and trailing chromatin as a function of spindle length (SPB-SPB distance, n> 40 for each category). E. Spindle lengths in cells showing chromatin bridges. Boxes indicate 25th, median and 75th percentile. Whiskers are the minimum and maximum (n>50). *** P<0.001.
LNA-miR-29 increases collagen in murine plaques.Figure 3ApoE-/- common carotid arteries (CCA) were dissected into plaque and adjacent non-plaque areas, then RNA isolated. (A) Graphed qPCR quantification of collagen 1A1 (left panel) and collagen 3A1 (right panel) mRNA expression in plaque (P) and non-plaque (NP) areas of ApoE-/- carotid arteries. Three assays were carried out, and the data represent mRNA expression normalized to NP, and the mean + SEM, n=4, 3, 4 of mice/group. * represents p<0.001 compared with LNA-miR-29 non-plaque region. Picrosirius red staining of collagen in BCs (10x magnification) (B); brightfield (upper panel) and polarized light (lower panel) imaging shown. Collagen quantification in BCs(C, *p=0.016) from polarized light images expressed as a percentage of plaque area. Picrosirius red staining of collagen in aortic roots (4x magnification) (D); brightfield (upper panel) and polarized light (lower panel) imaging shown. Collagen quantification in roots (E, *p=0.003, #p=0.005) from polarized light images expressed as a percentage of plaque area.
In vivo analysis of autophagic vesicles, inflammation, and cell death in infected mice with Mtb-Δeis.Figure 6C57BL/6 mice were challenged, by aerosol, with 10-30 CFU Mtb-WT, Mtb-Δeis, or Mtb-c-eis and sacrificed 4 weeks post-infection. (A) High- and low-magnification electron micrographs of lung tissue sections from mice infected with Mtb-Δeis show accumulation of autophagic vesicles (black arrow, bacteria in autophagic vacuoles; white arrow, degradative autophagic vacuoles). Scale bars: 2 µm (left upper), 0.5 µm (right). Numbers of autophagic vacuoles per cell in each TEM section (left lower) (mean±SD; n = 50). (B) Quantitative RT-PCR analysis of lung tissue from Mtb-WT-, Mtb-Δeis-, and Mtb-c-eis-infected mice. Total RNA was extracted from paraffin-embedded lung tissue sections, as described in the Materials and Methods. (C) To assess in vivo cell death, bronchoalveolar lavage fluid cells from Mtb-WT-, Mtb-Δeis-, and Mtb-c-eis-infected mice were subjected to PI staining, and analyzed by flow cytometry. Data are presented as the mean±SEM (n = 4). (D) Numbers of CFUs in lung and spleen 4 weeks after infection with Mtb-WT, Mtb-Δeis, or Mtb-c-eis. Data are presented as log10 CFU±SEM (n = 4). (E) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis and then analyzed by CFU assay. CFU data represent the mean±SD of four individual experiments. **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition. UI, uninfected.
Fig.Figure 3HE, Oil Red O (ORO), Cytochrome c Oxidase (COX) and Succinate Deshydrogenase (SDH) staining of representative frozen sections of TA muscle from 3-month-old HSA-Cre and control mice. Quantification of triacylglycerol (TAG) levels in GC muscles from 3-month-old HSA-Cre and and control mice. Content of TAG assessed by MS in HSA-Cre and control GC muscles. Cholesterol, (E) Cholesteryl ester (CE), (F) DAG was assessed by MS in HSA-Cre and control GC muscles.
Effects of FUNDC1, HSC70, HSP70, TOM complex, Bcl-xL and LONP1 on the formation of MAPAs in response to proteasome inhibition.Figure 4HeLa cells were transfected with GFP-p62, treated with DMSO or 10 μM MG132 for 8 hours and then immunostained with anti-Ub and anti-γ-Tubulin antibodies. Note that the aggresome (blue arrowhead), rather than the MAPAs (white arrowheads), colocalizes with γ-Tubulin. Scale bar=10 µm. B, C. HeLa cells were stably transfected with the indicated vectors, treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm. B, C. HeLa cells were stably transfected with the indicated vectors, treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm. Quantification is show in (C). D, E. HeLa cells were transfected with Ctrl, TOM20, TOM22 or TOM70 shRNA vectors including nuclear localized GFP (N-GFP) expressing elements, then treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm. Quantification is show in (E). F, G. HeLa cells were transfected with GFP or GFP-Bcl-xL, then treated with MG132 and immunostained with anti-p62 antibodies. Scale bar=10 µm. Quantification is show in (G). H, I. HeLa cells were stably transfected with Ctrl or LONP1 shRNA, treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm. Quantification is show in (I).
Figure 6Growth assay of wildtype, ∆Gid2, ∆Art2, and Art2P2S yeast strains on SD-Lys (-) and SD-Lys containing 1.0 μg/mL thialysine (+). Spots represent 1:2.6 serial dilutions. Growth assay of wildtype yeast or yeast strains containing the indicated deletions on SD-Lys (-) and SD-Lys containing 1.5 μg/mL thialysine (+). Spots represent 1:5 serial dilutions. Growth assay of wildtype yeast or yeast strains containing the indicated deletions on SD-Lys (-) and SD-Lys containing 1.5 μg/mL thialysine (+). Spots represent 1:5 serial dilutions. Growth assay of wildtype and ∆Art1 strains containing GID2 or GID5 deletions on SD-Lys (-) and SD-Lys containing 1.0 μg/mL thialysine (+). Spots represent 1:5 serial dilutions. Growth assay of wildtype and ∆Art1 strains containing GID10 or GID4 deletions on SD-Lys (-) and SD-Lys containing 1.0 μg/mL thialysine (+). Spots represent 1:2.6 serial dilutions. Growth assay of wildtype and Rsp5F618A strains containing GID2 or GID5 deletions on SD-Lys (-) and SD-Lys containing 0.75 μg/mL thialysine (+). Spots represent 1:2.6 serial dilutions. Growth assay of wildtype and Rsp5W415F,P418A (Rsp5WW3*) strains containing GID2 or GID5 deletions on SD-Lys (-) and SD-Lys containing 0.5 μg/mL thialysine (+). Spots represent 1:2.6 serial dilutions. Growth assay of wildtype and Art2P748A,P749A,Y750A (Art2PPY*) strains containing GID2 or GID5 deletions on SD-Lys (-) and SD-Lys containing 1.0 μg/mL thialysine (+). Spots represent 1:2.6 serial dilutions.
Figure 5Apical view of wing imaginal disc with homozygous Dl∆β1-2 clones (GFP negative) stained for Dl (red) and Cadherin (blue). (A') Z-projection of apical layers spanning a Dl∆β1-2 clone (GFP negative) located at the DV boundary. No change in apical localisation of Dl (red) and Cadherin (blue) is detected. Panels on the right show the Dl (grayscale) apical localisation on the wing imaginal disc. Uptake assay at t=0. After exposure to extracellular anti-Dl antibody, Dl protein (red) is detected at similar levels apical to Cadherin (blue) in wild-type (GFP) and Dl∆β1-2 mutant tissue (GFP negative). (B') Cross-sectional view of B, Dl protein (red) is present apically relative to Cadherin (Blue) which marks adherens junctions. Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin (Cad) and Delta (Dl), respectively. Uptake assay after 30 minutes, internalised anti-Dl (red) enters the endocytic route and in the cross sectional view (C') can similarly be detected as puncta along the cell axis in wild-type (GFP) and homozygous Dl∆β1-2 (GFP negative) tissue. Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin (Cad) and Delta (Dl), respectively.
Prom1 is expressed in mouse primary hepatocytes and its deficiency prevents glucagon-induced gluconeogenesis A-I.Figure 1A-I. Correlative light electron microscopy (CLEM) showing Prom1 at the plasma membrane of primary hepatocytes. Primary hepatocytes were isolated from 12-week-old male WT (Prom1+/+) or KO ( Prom1cre-ert2-nlacz) mice. The boxed areas in panel (A, D, G) and (B, E, H) were magnified and presented as panel (B, E, H) and (C, F, I), respectively. Scale bar; 5 μm (A, D and G), 1 μm (B, E and H), and 0.2 μm (C, F and I). J. Immunofluorescent staining of Prom1, CK19 and HNF4α in two serial cryosections of mouse liver. The boxed area in the top panel was magnified and presented as the bottom panel. Asterisks (*) in the top panel indicate cholangiocytes of the bile bucts and triangles (Γ) in the bottom panel indicate hepatocytes. Scale bar, 100 μm. Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (K) Differentially expressed genes showing more than a 2-fold change with P < 0.05 (118 DEGs) induced (n=55) or repressed (n=63) by Prom1 knockout (n = 2/group). G6pc, glucose-6-phosphatase catalytic subunit; Pck1, phosphoenolpyruvate carboxykinase 1. Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (L) Relative expression levels of G6pc and Pck1 mRNAs after serum starvation for 16 hours were determined by qRT-PCR (n = 3/group). Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (M) Levels of the Prom1, PCK, G6PC and GAPDH proteins were determined by immunoblotting. Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (N) Prom1+/+ or Prom1-/- hepatocytes serum-starved for 8 hours were incubated with 2-[3H]deoxyglucose for 10 minutes. Glucose uptake was assessed by calculating the amount of cell-associated radioactivity normalized to the amount of protein (n = 3 mice/group).
Defective O-GlcNAc signaling in intestinal epithelial cells in IBD patients.Figure 1A, B Representative images of OG immunohistochemistry on paraffin-embedded colon sections from control, UC and CD patients (n = 4). Intensities of staining were scored on the right. Scale bars = 50 μm. (one-way ANOVA, OGT: Healthy vs UC P=0.0002, Healthy vs CD P=0.003; O-GlcNAc: Healthy vs UC P=0.0365, Healthy vs CD P=0.0125  A, B Representative images o O-GlcNAc (B) immunohistochemistry on paraffin-embedded colon sections from control, UC and CD patients (n = 4). Intensities of staining were scored on the right. Scale bars = 50 μm. (one-way ANOVA, OGT: Healthy vs UC P=0.0002, Healthy vs CD P=0.003; O-GlcNAc: Healthy vs UC P=0.0365, Healthy vs CD P=0.0125  C Levels of intestinal epithelial OGT and O-GlcNAcylation in a second cohort of control, UC, and CD patients. (one-way ANOVA, OGT: Control n=10, UC n=9, CD n=6; Control vs UC P=0.035, Control vs CD P=0.003; O-GlcNAc: Control n=10, UC n=8, CD n=8; Control vs UC P=0.0027, Control vs CD P=0.008  D, E Regression plots of average immune-staining scores of OGT and O-GlcNAcylation against histological scores in UC (D) and CD (E) (D: OGT n=19, O-GlcNAc n=18; E: OGT n=15, O-GlcNAc n=16)
Figure 2Immunofluorescence shows that the Vret signals (green) co-localize with the Ago3 signals (red). DAPI shows the nuclei (blue). Scale bar: 10 μm. The three panels on the right-hand side show high magnification images of the part indicated by the white dashed line box in the image on the left-hand side. This panel arrangement applies also for panels B−H. The bar chart shows the percentage of Vret-positive nuage in Ago3-positive nuage. Immunofluorescence shows that the Vret signals (green) partially co-localize with the Siwi signals (red). DAPI shows the nuclei (blue). Scale bar: 10 μm. The bar chart shows the percentage of Vret-positive nuage in Siwi-positive nuage. Immunofluorescence showing the Ago3 signals (green) in control and Vret-depleted BmN4 cells (Vret KD). Vret is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing the Siwi signals (green) in control and Vret-depleted BmN4 cells (Vret KD). Vret is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing the Vret signals (red) in control and Ago3-depleted BmN4 cells (Ago3 KD). Ago3 is shown in green. DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing the Siwi signals (red) in control and Ago3-depleted BmN4 cells (Ago3 KD). Ago3 is shown in green. DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing Flag-Ago3 WT, KA mutant and DDH mutant signals (green) expressed in naïve BmN4 cells (Control). DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing Flag-Ago3 WT, KA mutant and DDH mutant signals (green) in Ago3-depleted cells (Ago3 KD). DAPI shows the nuclei (blue). Scale bar: 10 μm.
Figure 5Reduction in the number of formed spheroids (optical microscope) generated by 1x106 disaggregated cells (cultured in stem cell conditioned media and low-adhesion plates) obtained from CXCR4+ M5 subcutaneous tumors, 24h after 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. (N=8; 2 mice/group; 4 plates/mouse). (Scale bar, 100µm). Reduction in tumor re-initiation capacity after subcutaneous inoculation of 5x106 cells in NSG mice (N=4 tumors/group) derived from disaggregated tumor cells obtained from SC tumors 10 days after administration of 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days , as compared to Free oligo-FdU-treated or Buffer-treated mice. Recording of the number and size of positive tumors. (N=4; 2 mice/group, 2 injection points/mouse). T22-GFP-H6-FdU-induced reduction in the number of intravasated tumor emboli in peri-tumoral vessels of the M5-orthotopic primary tumor (E. optical images; F. emboli number quantitation) and reduction of CXCR4 expression in these emboli (G), treated 7 days after tumor cell implantation with 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. Tumor emboli counting in 10 high powder field at 200x magnification in H&E stained sections from each tumor. (N=5/group).
Figure 3(A) Representative immunoblot of Cand2 protein levels in mouse organs. (B) Cand2 expression in mouse organs measured by RT-qPCR. n=3 biological replicates. (C) Immunofluorescent staining of paraffin sections from hearts from WT and Cand2 KO mice. CAND2 (stained in red), ACTIN (stained in green) and nuclei (stained with DAPI, blue) in. Scale bar 20μm (D) Representative immunoblot of Cand2 protein from different cardiac cell types CM- cardiomyocytes, EC - endothelial cells, FB - fibroblast. (E) Immunofluorescence of adult rat cardiomyocytes of Cand2 (stained in red), sarcomeric actin (stained in green), and nuclei (stained with DAPI blue). Scale bar 50μm. (F) Immunoblot of subcellular fractionation of left ventricle (LV). Gapdh and Histone 3D (H3D) used as cytoplasmic and nuclear markers, respectively. WCL - whole cell lysate, cyt - cytoplasmic fraction, nuc - nuclear fraction.
Figure 8In vitro incubation of one of the Alu RNA consensus sequences for different incubation periods. In vitro transcribed Alu RNA (67nM) incubated at 37oC with 250nM HSF1 in the course of 6 hours with time intervals of 1 hour. Comparison among Hsf1 (~60KDa), denatured HSF1, Poly A polymerase (~56KDa) and no protein (just TAP buffer) with regard to Alu RNA processing (estimating relative full length RNA remaining) (two replicates). The full gels are available as Source Data For Figure 8. Relative full length RNA remaining was calculated using ImageJ area under the curve software over time. Error bars represent standard deviation from the mean. Comparison among Alu RNA (three replicates), and two control RNAs (two replicates) regarding the full-length RNA levels remaining after in vitro incubation for 90 minutes at 37C with HSF1. Sizes of control RNAs are control for RNA #1, 143nt and for control RNA #2, 432nt. Incubation in the absence of HSF1 but presence of the same buffer (TAP) was used as control to take into account any non HSF1 specific RNA destabilization due to non-specific degradation. Threshold for statistical significance was a p value of 0.05 with p=0.03 (unpaired, non-directional t-test) for the comparison between HSF1 and no protein incubation (n=3, for Alu RNA). The full gels are available as Source Data For Figure 8. Error bars represent standard deviation from the mean. Plotting of the position of the first base (5' end) of Alu RNA fragments across the Alu consensus sequence produced by Alu RNA that has been processed in vitro for 90 minutes at 37C in the presence of HSF1 (upper panel) and compared with one of the in vivo samples of Figure 1 (lower panel). The x axis represents an Alu RNA metagene aligned at the start site of the Alu consensus sequence and the y axis shows the 5' end count for Alu RNA fragments aligning to any position downstream of position +1. The in vivo sample x axis depicts a 11nt sift compared to the in vitro one. Scatterplot depicting the positive correlation between HSF1 mRNA expression values and Alu RNA processing ratio in MAP AD patients (r=0.72, p<0.001). Statistical test is based on Pearson's product moment correlation coefficient and follows a t distribution using the cor.test function in R package stats. Scatterplot depicting lack of correlation between HSF1 mRNA expression values and full length Alu RNA levels in MAP AD patients (ns=non-significant, with p value 0.05 as the significance threshold). Statistical test as in subfigure 8E.