EMBO/sd-character-level-ner huggingface dataset:
Loss of MALAT1 expression in rod photoreceptors with longer post-mortem time.Figure 4Fluorescent in situ hybridization analysis of the same donor human peripheral retina at different time points post-mortem (7 and 13 hours, Retina 7), showing decreases in MALAT1-hi rod subpopulations in the outer nuclear layer (ONL) at later time point. INL: inner nuclear layer; OPL: outer plexiform layer. Scale bar = 20µm. White arrows indicated MALAT1-hi rod photoreceptors.
The differential effects of AMPK agonists correlates with the expression of inducible nitric oxide synthase (iNOS).Figure 4
a) Bif-1+/+ and Bif-1−/− MEFs were cultured in complete medium (CM) or EBSS for 3 h and analysed by transmission electron microscopy.figf3(a) Bif-1+/+ and Bif-1−/− MEFs were cultured in complete medium (CM) or EBSS for 3 h and analysed by transmission electron microscopy. Arrows indicate autophagosomes. The number of autophagosomes per cross-sectioned cell was counted (mean ± s.d.; n = 42). (b) Bif-1+/+ and Bif-1−/− MEFs were transfected with GFP-LC3, cultured in LC3 or complete medium for 3 h, then analysed by fluorescent microscopy. The number of GFP-LC3 dots per GFP-positive cell was counted (mean ± s.d.; n = 35). (c) Bif-1+/+ and Bif-1−/− MEFs were cultured in EBSS for the indicated times and subjected to immunoblot analysis with anti-LC3, anti-α-tubulin and anti-β-actin antibodies. (d) Bif-1−/− MEFs stably transfected with a Bif-1 expression plasmid or control empty vector were cultured in EBSS for 0, 3 or 6 h and subjected to immunoblot analysis. The immunoblotting results of LC3 in c and d were quantified as the percentage of LC3-II out of total LC3 (LC3-I + LC3-II) and are represented in bar graphs. (e) Stable Bif-1 shRNA-expressing clones or control HeLa cells transfected with GFP-LC3 were cultured in complete medium or EBSS for 2 h and analysed by fluorescent microscopy. The number of GFP-LC3 dots per GFP-positive cell was quantified (mean ± s.d.; n = 66). (f) Bif-1 knockdown HeLa cells were transfected with GFP-LC3 in combination with Bif-1 shRNA-resistant Bif-1-HcRed, Bif-1ΔBAR-HcRed or empty HcRed vector for 20 h. The cells were then cultured in EBSS or complete medium for 2 h and the number of GFP-LC3 dots per HcRed-positive cell was determined using a fluorescence microscope (mean ± s.d.; n = 35).
EGF induces rapid and transient deSUMOylation of transcriptional regulators in HeLa cells.Figure 1(A) Schematic representation of a quantitative proteome analysis that compares the SUMO proteome of serum-starved HeLa cells treated for 10 min with or without 100 nM EGF (SILAC labeling and endogenous SUMO1- and SUMO2/3-IPs). (B) Scatterplots represent SILAC/SUMO-IP quantification after EGF treatment from three biological replicates. Each dot represents a protein that is either present in the same ratio between untreated and EGF treated cells (dark blue dots) or is significantly more present in one of the samples (red and yellow dots). (C) Left panel: Bar graph depicting mass spectrometry results of 11 proteins with altered SUMOylation (significant hits with P<0.0001), as well as the three non-changing proteins SUMO2, RanGAP1 and TRIM28. Right panel: Proteins highlighted in bold in the left panel were validated by SUMO-IP/immunoblotting from serum starved HeLa cells without or with 10 min EGF stimulation. (D) Time course experiment: serum starved HeLa cells were treated with 100 nM EGF, samples were harvested at indicated times and subjected to SUMO2 IP followed by immunoblotting with the indicated antibodies. IRF2BP1, IRF2BP2 and TRIM24 are rapidly but transiently deSUMOylated upon EGF treatment.
Cellular phenotypes mediated by pyk1 allele replacement.Figure 5(A) Spot assays on solid media from a three-fold dilution series of exponential cultures at the same cell density in 96-well plates (3 biological replicates of each strain) and spotted in 16 technical replicates (each dilution in 4x4 square). The A-strain is more resistant to antimycin A but less resistant to oxidative stress triggered by H2O2 or diamide. A control without toxin (left) was included in each batch of spot assays performed and a representative image is shown here. (B) Fitness (approximated by maximum slope of smoothed growth curves) of A- and T-strains on 12 carbon sources, with either yeast extract (YE) or ammonium (NH4), with or without 0.1% priming glucose to support initial growth. For all 48 conditions, two biological replicates of A- and T-strains were grown in technical quadruplicates each. Dotted lines in panels mark a fitness ratio of 1 (i.e., same fitness). (C) Fitness of A- and T-strains on 95 nitrogen sources on Biolog Phenotype MicroArrays. Conditions with no substantial growth were excluded (black circles, maximum slope <0.015). Red lines show arbitrary significance cut-off, put at |log2(A-strain/T-strain)|>0.75. (D) Fitness of A- and T-strains on 72 different drugs and toxins, at 4 concentrations each, on Biolog Phenotype MicroArrays. (Results for benzamidine were inconclusive, with both strains comparatively resistant in one concentration each.) (E) Spot assays to validate selected results Several assays were performed with both h- and h90 strains, and no mating-type specific differences were evident between the two sets of allele-swap strains. (F) Chronological lifespan of A- and T-strains, i.e. the proportion of non-dividing cells in stationary phase that maintain proliferative potential after refeeding. The data show colony forming units (CFUs) per ml of culture over 7 days of stationary phase in glucose-depleted rich media. Three biological repeats were carried out for both strains, with each repeat measured as technical triplicates. Error bars represent standard error of the biological replicates. (G) Boxplot showing resistance to 3mM H2O2 grouped by pyk1 allele for 156 strains from our collection. The T-strains had a higher mean fitness in H2O2 than the A-strains (1.01±0.06 vs 0.95±0.11; p=0.0021, Welch's t-test). The resistance score was obtained as for antimycin A. (H) Boxplot showing growth fitness on rich media, grouped by pyk1 allele for 158 S. pombe strains (0.88±0.16 vs 0.94±0.24; p=0.18, Welch's t-test).
Figure 2A In vivo suppressive function of WT and Traf6fl/flFoxp3Cre+ Tregs. Tregs from the indicated mice (CD45.2+) were isolated by FACS, as were naïve (CD62Lhigh/CD25-) CD4+ responder T cells from congenically distinct (CD45.1+) donor mice. Tregs and Tresponders were mixed at a 1:5 (2x105:10x105) ratio before injection into Rag2-/- mice. 7 days later, spleens were harvested, and the relative frequencies and absolute numbers of each transferred cell populations were determined by flow cytometry. B Implanted B16 melanoma growth in Traf6fl/flFoxp3Cre+ and wild type (Traf6fl/flFoxp3Cre-) mice. 1x105 B16F10 cells were injected subcutaneously (s.c.) into the shaved flanks of the indicated mice (n= 5/group). Tumor volumes were monitored every 3 days. C, D Proinflammatory cytokine production in tumor-bearing mice with and without Treg-specific TRAF6 expression. Cell suspensions of the tumor-draining lymph node and tumor-infiltrating leukocytes (TILs) were recovered after 21 days of tumor growth. Ex vivo stimulation with PMA and ionomycin in the presence of Golgistop for 5 hour preceded intracellular staining for IFNγ and IL-17 and flow cytometry analysis. E, F FOXP3 expression by CD4+ T cells in tumor bearing Traf6fl/flFoxp3Cre+ and wild type (Traf6fl/flFoxp3Cre-) mice. The frequency of FOXP3+ cells in the CD4+ cells of tumor-draining lymph nodes (dLN), peripheral, non-tumor draining lymph nodes (pLN), spleens, and TILs were determined by flow cytometry.
Figure 7In mature schizonts, SERA6 and its essential protein cofactor, MSA180 are initially spatially separated from SUB1. Activation of the kinase PKG triggers discharge of SUB1 into the PV lumen where SUB1 initiates the coordinated maturation of both SERA6 and MSA180 by proteolysis, culminating in the formation of an active SERA6-MSA180 protease complex, perhaps in the form of multimeric aggregates. PVM rupture allows the active SERA6-MSA180 complex to access the RBC cytoskeleton, where it cleaves β-spectrin, detaching the spectrin network from the RBCM. This rapidly leads to RBCM rupture and merozoite egress.
PDGFR-β expression is upregulated in fibrotic human and murine kidneys (A) Immunoblot detection and its quantification of PDGFR-β in kidney cortex lysates of healthy and diseased human kidneys shows twice as much PDGFR-β abundance in diseased tissue.Figure 1Immunoblot detection and its quantification of PDGFR-β in kidney cortex lysates of healthy and diseased human kidneys shows twice as much PDGFR-β abundance in diseased tissue. A stronger signal for phosphorylated PDGFR-β indicates the active status of the receptor. The high α-SMA content in the diseased kidneys shows that they are affected by fibrosis. Bar graphs show means ± SD, healthy n = 5, diseased n = 6 PDGFR-β staining is detectable in mesangium of healthy (a) and diseased (b-d) glomeruli of human kidneys. PDGFR-β positive mesangial cells expand in mesangioproliferating and scarring glomerular changes. Similarly, the population of PDGFR-β positive interstitial cells is discrete in healthy kidney cortex (e) but expands in fibrosis (f-h). Arrows point to selected areas of positive PDGFR-β staining. Scale bar = 50 µm GFP expression under Pdgfrb-promoter in mice is present in mesangial cells of glomeruli and interstitial cells of cortex and medulla, but not in tubular cells. Nuclei are stained with DAPI (blue). Scale bar = 50 µm Tissue clearing with Scale and 3D reconstruction of Pdgfrb-GFP reporter mice in a healthy and fibrotic kidney (UUO day 5) shows the expansion of Pdgfrb expressing cells in fibrosis.
Figure 5A Location of gRNAs flanking the NF-κB binding site (highlighted). B Primary CD4 T cell editing efficiency for indicated combinations of 5' and 3' gRNAs (n=6 for DB, DH, FH, and n=2 for FB). Distribution of indels assessed using ICE. C Experimental workflow: equimolar amounts of 5' and 3' gRNA-containing RNPs (fluorescently tagged with ATTO-550) were nucleofected into resting CD4 T cells, which were stimulated after 48 hours with anti-CD2/3/28 microbeads and IL-2. D Expression of genes on 6q23 in EU-containing mRNA (EU added at time of stimulation) showing that deletion of the NF-κB binding site specifically reduces transcription of TNFAIP3, but not other genes at this locus (n=6; one sample t-test). Representative data from the DH gRNA combination. E Expression of CD69, an activation marker, following CRISPR editing with indicated gRNA combinations or the non-targeting (negative) control (NTC) - data shown for ATTO-550 positive (RNP-containing) and negative cells (n=6; paired t-test, one-tailed). Inset flow cytometry plot depicting representative gating of ATTO-550 positive and negative cells. F Correlation between editing efficiency (total indel rate) and levels of phosphorylated IκBα in CD4 T cells (normalised to the mean fluorescence intensity in NTC; n=9, linear regression). G Secretion of effector cytokines following deletion of the NF-κB binding site - Th1 (left panel, IFNγ), Th17 (centre panel, IL-17A) and Th2 subsets (right panel, IL-4) (n=6, paired t-test, one-tailed).
Figure 3(A) Representative images of single-cell alkaline gel electrophoresis (comet assay) of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). (B) Comet tail moment from single-cell alkaline gel electrophoresis (comet assay) of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). More than 250 total cells were considered. The mean ± SEM of the median from at least three independent experiments is shown, except for RAD1, RAD17 and UBE2B (n=2). *p<0.05, **p<0.01, ***p<0.001 (one-tailed unpaired t-test). Black stars denote significant increases whereas red stars denote significant decreases.
Figure 1(A) Phenotype appearance of the Gmlmm1-1 mutant. Typical leaves (left panel; scale bar, 1 cm) and whole plants (right panel; scale bar, 10 cm.) were photographed 60 and 118 days after seeding, respectively. Data information: The experiments were performed three times as biological replicates, with similar results. (B) Enhanced resistance of Gmlmm1-1 and Gmlmm1-2 to Psg infection. Leaves from 2-week-old soybean plants were infected with Psg and the bacteria number was determined at 0 and 4 dpi (Mean ± SD, n = 8, n represents sample number, ***, p < 0.001, Student's t-test). Data information: The experiments were performed three times , as biological replicates, with similar results. (C) Physical locations of markers defining the GmLMM1 region. The chromosomal positions (Pd, physical distance in Mb) of each marker are indicated on chromosome 13 (Chr13) according to the soybean reference genome. Recombination ratio (Rr, %) at the corresponding physical location was calculated using a formula (2 × plant number of genotype HD 12 + plant number of heterozygote genotype) / (2 × total number of plants) for rough mapping. Numbers of recombination (Nr) refers to the number of heterozygous plants at a physical location in the 92 plants for gene mapping. The candidate genes were harbored in a 131 kb region spanned by MOL3754 and MOL3780, in which six homologous genes were tandemly repeated and belonged to the malectin-like gene family in the shadows. The direction of the arrow represents the direction of the gene on the chromosome. Genes with high similarity are indicated by arrows of the same color. The number under the genes corresponds to the gene locus on chromosome 13. The Glyma.13G054400 (44) gene harbors a mutation (C to T) according to re-sequencing. (D) Comparison of Arabidopsis FERONIA, ANXUR1 and GmLMM1. SP and TM indicate the predicted signal peptide and transmembrane domain, respectively. Two corresponding mutated sites in Gmlmm1-1 and Gmlmm1-2 are indicated by arrows. (E) Subcellular localization of GmLMM1-GFP and GmLMM1L407H-GFP in N. benthamiana. PI4P indicates the membrane localization. Scale bar, 10 μm. GFP, green fluorescent protein. Data information: The experiments were performed three times as biological replicates, with similar results. (F) Autophosphorylation activity of the GmLMM1 intracellular domain in vitro. The proteins purified from E. coli were subjected to pMIAGO phosphorylation detection assay (upper panel) and were detected using western blot (lower panel) with BIK1-HIS as a control. GmLMM1K564E contains a mutation at the predicted ATP binding site. Data information: The experiments were performed two times (F), as biological replicates, with similar results.
Germline autophagy mutant egg chambers exhibit reduced expression of cleaved caspase-3.Figure 3.Germline autophagy mutant egg chambers exhibit reduced expression of cleaved caspase-3. Confocal micrographs of stage 13/14 egg chambers stained for cleaved caspase-3(casp-3). (A) Wild-type (wt) late stage 13 egg chamber showing cleaved caspase-3 staining in the degenerating nurse cell cluster. (B) In wild-type stage 14 egg chamber, nurse cells are completely degraded (arrows). (C-E) atg1−/− (C), atg13−/− (D), and vps34−/− (E) germline mutant stage 14 egg chambers exhibit significantly reduced staining for cleaved caspase-3 and contain persisting nurse cell nuclei. Nurse cells are outlined with a white line. Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. (F) Quantification of mean intensity of cleaved caspase-3 staining observed in the nurse cells of stage 12-14 egg chambers in wild-type and germline autophagy mutant egg chambers. Wild type (WT): three independent experiments, n = 30 egg chambers; atg1−/− GLCs: four independent experiments, n = 30 egg chambers; atg13−/− GLCs: four independent experiments, n = 30 egg chambers; vps34−/− GLCs: three independent experiments, n = 30 egg chambers. Data are presented as mean ± SD. Difference was significant with P < 0.001 for all values versus wild type. Bars, 20 µm.
Figure 1Scheme of the E. coli division elements involved in the formation of condensates. The concentrations of FtsZ, SlmA and SBS used in this study were 12, 5 and 1 μM, unless indicated otherwise. Representative confocal images of the FtsZ·SlmA·SBS condensates formed in 150 g/L dextran 500 or Ficoll 70, and absence of condensates in dilute solution. Scale bars: 20 μm, except for images at higher magnification in dextran (5 μm, second row). Turbidity of FtsZ·SlmA·SBS in buffer (n = 3), in 150 g/L dextran 500 (n = 5) or Ficoll 70 (n = 3) and in 50 g/L PEG 8 (n = 3). Data correspond to the average ± S.D.
H19 stimulates proliferation of endometrial stromal cells.Figure 3A-F The indicated cells were transfected with siCon, siH19, empty vector, or pH19. RNA, cell viability (as indicated by viable cell numbers), and caspase activity (as a readout for apoptosis) were analyzed 48 h post-transfection. Combined results from two patient cells in each group are presented. G E2 stimulates H19 expression in endometrial stromal cells. Endometrial stromal cells #166 and #98 were stimulated with E2 (+) or vehicle (−) for 48 h, followed by RT-qPCR analysis of RNA extracted from the cells. Results combining two patient cells are shown. H A proposed model of the H19/let-7/IGF1R-mediated regulation of endometrial stromal cell proliferation. During the proliferative phase of the endometrium, E2 stimulates the expression of H19. As its level rises, H19 acts as a sponge to sequester let-7 and prevent it from inhibiting its target gene Igf1r. Increased IGF1R protein level leads to increased IGF1 signaling with a biological endpoint of increased proliferation of endometrial stromal cells.
Distinct binding distributions of HIF-1 and HIF-2 are conserved across cell types.Figure 6Canonical HIF-1 and HIF-2 binding sites were determined by overlap of HIF-α and HIF-1β binding sites identified by the MACS peak caller as for Figure 4. The distance from each binding site to the nearest annotated gene promoter was determined and plotted as a frequency distribution for (A) HIF-1 and (B) HIF-2 sites in HKC-8 cells, (C) HIF-1 and (D) HIF-2 sites in RCC4 cells, (E) HIF-1 and (F) HIF-2 sites in HepG2 cells. The distribution of HIF-1 binding sites was consistently more promoter proximal than HIF-2 sites despite the sites themselves differing between cell types The distribution of accessible HRE motifs was also plotted in HepG2 cells and differed significantly from that of both HIF-1 (p~10-50) and HIF-2 (p~10-80) binding sites (Chi-squared test)
Figure 7Western blot showing HNF1β over-expression after lentiviral transduction of PANC1 cells. Expression of FOXA2 in wild type and over-expressing (O.E.) cells is shown. Vinculin: loading control. Scatter plot showing the genomic distribution of FOXA2 in PANC1 cells transduced with the HNF1β-expressing lentivirus (HNF1B O.E.) and in their matched mock-infected control. The fraction of gained, unchanged and lost FOXA2 peaks overlapping HNF1β ChIP-seq peaks in transduced PANC1 cells is shown in the pie-charts on the right. TF motif over-represented in gained or lost FOXA2-bound genomic regions. Representative snapshot from PANC1 cells transduced with the HNF1β-expressing lentivirus. HNF1B O.E.: HNF1B over-expression.
Figure 6Surgical specimens were collected from 72 non-small cells lung cancer (NSCLC) patients. In (A) age at surgery and sex distribution of the patients are reported. Data are depicted as a box plot. The central band represents the mean ± quartiles, the whiskers mark the range. (B) Copy number (CN) variations in GOLPH3 gene were assessed in cancer tissue section by DNA FISH as described in Materials and Methods and are expressed as a pie chart. (C) Cancer tissue sections were processed for IHC using antibodies against endogenous GOLPH3 and LCS. The IHC intensity of the staining for each section and for both the tested proteins was classified according to the criteria described in Materials and Methods. D. Representative images of NSCLC sections stained for GOLPH3 and LCS. Scale bar, 100 µm.
Figure 5Analysis of A549 cells with NLS-RL-WT or NLS-RL-CM either mock transfected or treated with poly(I:C) (PIC) for 5 hours. A. FISH analysis to detect poly(A)+ RNA and IF analysis against FUS protein. Images are a single 0.2 micron Z section. Analysis of A549 cells with NLS-RL-WT or NLS-RL-CM either mock transfected or treated with poly(I:C) (PIC) for 5 hours. B. Graph depicting the fraction of nuclei with FUS dispersed in the nucleoplasm or in foci in cells with reduced nuclear oligo(dT) signal. All the nuclei (total) in which nuclear RNA was degraded (N=64) or the same nuclei classified as to whether or not they contained residual poly(A) foci. Nuclei with poly(A) foci (N=14). Nuclei without poly(A) foci (N=50). Analysis of A549 cells with NLS-RL-WT either mock transfected or treated with poly(I:C) (PIC) for 5 hours. C. FISH analysis to detect poly(A)+ RNA and IF analysis against mediator complex protein, MED1, to detect super-enhancer condensates. D. Number of MED1 foci in mock and PIC treated NLS-RL-WT cells. E. Average volume of individual MED1 foci in mock and PIC treated NLS-RL-WT cells. Analysis of A549 cells with NLS-RL-WT either mock transfected or treated with poly(I:C) (PIC) for 5 hours. F. FISH analysis to detect poly(A)+ RNA and IF analysis against BRD4, to detect super-enhancer condensates. G. Number of BRD4 foci in mock and PIC treated NLS-RL-WT cells. H. Average volume of individual BRD4 foci in mock and PIC treated NLS-RL-WT cells.
Btz controls NMJ size independently of the EJC.Figure 4confocal images of the NMJ on larval muscles 6 and 7 in segment A3, stained with anti-HRP (magenta) and anti-Dlg (green). (A) BG380-GAL4/+ control; (B) BG380-GAL4/+; UAS-btz RNAi/+; (C) BG380-GAL4/+; UAS-mago RNAi/+; (D) BG380-GAL4/+; UAS-eIF4AIII RNAi/+; confocal images of the NMJ on larval muscles 6 and 7 in segment A3, stained with anti-HRP (magenta) and anti-Dlg (green). /+; (E) btz2/+ control; (F) btz2/Df(3R) BSC497; (G) Btz-WT; btz2/Df(3R) BSC497; (H) Btz-HD; btz2/Df(3R) BSC497. Scale bars, 30 μm (shown in A for A-D and in E for E-H). (I) quantifications of the number of boutons normalized to muscle surface area (x1000) in the indicated genotypes. ****, p<0.0001; ***, p<0.001 by unpaired t-test or Mann-Whitney test. n=15 (BG380-GAL4), n=12 (BG380>btz RNAi; BG380>mago RNAi; BG380>eIF4AIII RNAi), n=16 (btz/+), n=13 (btz/Df), or n=10 (Btz-WT; btz/Df; Btz-HD; btz/Df). Error bars show mean ± SEM. NMJ size is reduced by knocking down btz in motor neurons, but slightly increased by knocking down mago or eIF4AIII. The size deficit in btz mutants is equally well rescued by the wild type or EJC interaction-defective btz transgenes.
CD3+ T cells are permissive to artificial MitoT (Mitoception) resulting in an increase of their mitochondrial content.Figure 2A Schematic representation of the experimental design of the mitoception protocol. B Confocal microscopy images of human CD3+ T cells with endogenous MT-labeled with MitoTracker Red CMXRos (white arrows, left panel) or after mitoception with MitoTracker Green labeled-MT from UC-MSCs (white asterisk, right panel). C FACS analysis of MitoT on CD3+ human T cells after mitoception with increasing amounts of UC-MSC MT corresponding to the same cell number ratios used in co-culture experiments (n=4 biological replicates, with 4 different donors of UC-MSCs). Graph shows mean ± SEM and statistical analysis by Student's t-test. D Representative transmission electron microscopy images of FACS-sorted human CD3+MitoTneg and CD3+MitoTpos cells following mitoception with UC-MSC MT. Red arrows show human mitochondria. Quantification of human MT in sorted CD3+ MitoTneg and MitoTpos cells after mitoception with UC-MSCs, by transmission electron microscopy (n=25 independent cells analyzed per group). Graph shows mean ± SEM and statistical analysis by Student's t-test.