EMBO/sd-character-level-ner huggingface dataset:
C/EBP is critical for the regulation of Foxp3 expression in vivo during iTreg generation A, B Flow cytometry of intracellular Foxp3 staining of mesenteric lymph node cells of Balb/c mice that received RAG2-/-DO11.Figure 3Flow cytometry of intracellular Foxp3 staining of mesenteric lymph node cells of Balb/c mice that received RAG2-/-DO11.10 CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPβ. (A) The recipients were immunized via intravenous injection of OVA323-339 peptide (20 ug) 1 and 3 d after transfer of transduced cells and analyzed 5 d after the first immunization. (B) The recipients were fed 1% OVA solution in drinking water for 5 consecutive days. Representative experiments are shown in the left panel and pooled data from 4 (A) or 3 (B) independent experiments with mean values are shown on the right. Dot plots are gated for CD4+KJ.1.26+GFP+ (top) or CD4+KJ.1.26+GFP- (bottom) and numbers indicate percent Foxp3+ cells in the gate. Statistical analysis was performed using one-way ANOVA. (*p<0.05, **p<0.01; ns, not significant
a) Expression of ATF-2::GFP in 5-FU-treated hermaphrodites fed control or RNAi targeting the indicated genes (scale bars, 20 μm).f5(a) Expression of ATF-2::GFP in 5-FU-treated hermaphrodites fed control or RNAi targeting the indicated genes (scale bars, 20 μm). (b) The number of animals harbouring embryos with excessive ATF-2::GFP expression was scored in at least 150 animals and given as the fraction (%) of the total. (c) N2 or msh-6(pk2504) embryos were continuously monitored under differential interference contrast microscopy (scale bars, 5 μm) from the three-cell stage until completion of the four-cell stage. Magnified images of representative nuclei are shown. (d) The fraction of embryos with visible, enlarged nucleoli (arrows) after 5-FU treatment was scored. Bar graphs represent mean±s.d. from three independent experiments.
SLO and NADase inhibit lysosomal fusion to GAS-containing autophagosomes in oropharyngeal keratinocytes.Figure 5SLO and NADase inhibit lysosomal fusion to GAS-containing autophagosomes in oropharyngeal keratinocytes.A. Confocal microscopy of keratinocytes infected with GAS strain 188, 188SLO-, or 188NADase- demonstrating the association of the lysosomal marker LAMP-1 (red) with GAS (blue) contained within EGFP-LC3 (green)-positive compartments at 1 h, 3 h, and 6 h post-infection. Scale bar = 10 µm. B. Quantification of the percent of GAS within EGFP-LC3-positive compartments that are co-localized with LAMP-1 at 1 h, 3 h, and 6 h. Data represent mean values from three independent experiments in which at least 100 intracellular GAS were quantified for each time point in each experiment.
Figure 4A Growth kinetic measurements of SM exposed to AMSIN or various antibiotics with known cellular targets. The peptide or the compounds used were 4×CL. VAN, vancomycin; RIF, rifampicin; KAN, kanamycin. OD600 were calculated as measured values subtracting the background value from broth medium (OD600 = 0.356). B Killing kinetic measurements of SM exposed to AMSIN or its linear version. The peptide dose used was 4×CL. C Effect of AMSIN on the SM membrane at a 10×CL dose. Propidium iodide, a dead cell stain that emits red fluorescence when bound to DNA, was used to monitor membrane damage by the compounds as a function of time. Vancomycin and Meucin-18 were used as negative and positive controls, respectively. D Scanning electron microscopic observation of AMSIN-induced BM deformation. The doses used were 10×CL for AMSIN and vancomycin. E Cytoplasmic accumulation of the soluble cell-wall precursor UMP in vancomycin-treated (blue line), AMSIN-treated (red dashed line) or not treated (green line) culture of MRSA P1374. Inset: UMP identified by UPLC-HRMS with singlely and doubly protonated m/z observed. F Water-soluble and water-insoluble cell wall precursors (UMP, Lipid I and Lipid II). In the pentapeptide (L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala), Ala, Glu and Lys residues are denoted by a dot in green, red and blue, respectively. The γ-D-glutamic acid and the pyrophosphate group pointed by two arrows are the target sites of defensins in UMP or Lipid II, and the C-terminal D-Ala-D-Ala residues marked by a single square bracket are the target sites of vancomycin in UMP or Lipid II. G The predicted complex structure showing that the γ- glutamate and pyrophosphate group (shown as a stick model) are involved in direct interactions of UMP/Lipid II with AMSIN (presented as a molecular surface model). H ESI-MS detecting the non-covalent complex between AMSIN and UMP. The quadruply protonated complex (m/z 1304.8) is observed. Inset: comparison of intensities of the complex between two ratios of UMP:AMSIN Here, AMSIN is abbreviated as AMS due to space limitation. I Sensorgrams of UMP binding to AMSIN. The peptide was covalently immobilized to CM5 via its amine groups, and UMP with different concentrations flowed by. The 6.250 μM analyte concentration analyzed in duplicate is shown by a yellow dotted line. J The dose-response unit curve of UMP binding to AMSIN, which is fitted by the BIAevaluation v2.0.1. software using 1:1 Langmuir binding model.
Figure 2(A). Interaction between neutrophils and opsonized (red) or non-opsonized (blue) GFP+ iRBCs by time in a "pulse-chase" experiment. The percentage of GFP+-neutrophils following 5 min of co-culture was set as 100%. (B). Model of how a low number of GFP+ neutrophils may be explained by the escape of GFP+ iRBCs (upper part) and how a high number of GFP+ neutrophils might be explained by the elimination (phagocytosis or extracellular killing) of GFP+ iRBCs (lower part) by the neutrophils. The escape of GFP+ iRBCs will allow parasites to propagation, while elimination will impair parasite propagation and cause reduced parasitemia of the culture. (C). Parasitemia (percentage %) of NF54 iRBCs cultures with or without daily addition of freshly isolated neutrophils. (D). Parasitemia (percentage %) of cultures of RBC infected by different P. falciparum lines (NF54, Dd2, SL, PfEMP1-KO). The parasites were kept in culture for six days in the presence (Neut.) or absence (Cont.) of neutrophils. (E). Percentage change in parasitemia following neutrophil challenge of RBCs infected by different parasite lines (NF54, Dd2, SL, PfEMP1-KO). (F). Flow cytometric analysis of the effect of neutrophil challenge on parasite cell cycle progression. Neutrophils were stained with an anti-CD11b antibody, while iRBCs were labeled with SYBR green. Gating for iRBCs were performed by gating for the SYBR green and CD11b negative population, excluding the neutrophils that were SYBR green negative, but CD11b positive. (G). Short term neutrophil killing of late-stage NF54-luciferase positive RBCs in the presence (red) or absence (blue) of catalase. (H). Parasitemia of cultures of iRBCs challenged with neutrophils in the presence or absence of catalase. (I). Short-term luciferase-based killing assay using neutrophils from three different donors in the presence or absence of 5 uM cytochalasin D.
Figure 2(A) Confocal microscopy image analysis of sEV fusion to MGC-803 cells. The MGC-803 (left side) and MKN-45 (right side) cells were treated with sEVs derived from MGC-803 cells (Con sEVs) or LSD1 knockout (KO) MGC-803 cells (KO sEVs) and stained with PKH26 for 12 h. Additionally, the cell membrane was stained with Dio, while the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Scale bar = 100 µm. (B) Immunofluorescence confocal microscopy analysis of LSD1 (green) in LSD1 KO MGC-803 cells (left panel) and LSD1 KO MKN-45 cells (right panel) incubated with 20 μg/mL Con sEVs and KO sEVs for 12 h. The cell membrane was stained with PKH26, while the nuclei were stained with DAPI. Scale bar = 100 µm.
Mif2 auto-inhibition depends on its Cse4 and DNA binding motifs.Figure 6Schematic introduction of Mif2wt and Mif2-∆sigAT in which its Cse4 and DNA binding motifs have been removed. Cross linked Lysin residues that connect the N-terminus with residues within the signature motif or AT hook of Mif2 in isolation are depicted in blue and were the basis to create the Mif2-∆sigAT mutant. Mif2Flag-wt or -∆sigAT directly from insect cell lysate was immobilized on M2 Flag Agarose, washed and incubated with Mtw1-Nnf1 complex (MN). After washing, bound complexes were eluted by adding 3xFlag peptide. Western blotting against the His-tag on Nnf1 confirmed increased binding of MN to Mif2-∆sigAT, already visible in the Coomassie stained SDS-PAGE gel. Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mif2-wt or Mif2-∆sigAT and MN alone and in combination at 10 µM concentration. All combinations were incubated for 1 h at 4°C prior to the run. MN (blue), Mif2 (red), combination (green). Note that the same MN elution profile and SDS-PAGE (upper panel) is displayed in both sets to improve clarity. Dashed boxes highlighting corresponding fractions were included to improve comparability.
GemC1-deficient mice die within the first postnatal week.Figure 5A. Schematic representation of the wild type (WT) and knock-out (KO) GemC1 alleles. A lacZ-neo cassette has been inserted between exons 2 and 3 of the GemC1 gene. A splicing acceptor site (SA) upstream of lacZ ablates gene function (knock-out first allele, [38]) B. GemC1KO/KO mice compared to their wild type littermates (GemC1KO/WT) at P5. C. Survival plot of mice homozygous for the GemC1 deletion (GemC1KO/KO, n=8) and control wild type or heterozygous littermates GemC1 (GemC1WT, n=14). GemC1 knock-out mice exhibit markedly reduced growth after birth and die within the first postnatal week.
Figure 4U2OS mCherry-LacR-Fok1 cells were treated with the indicated siRNA and subsequently transfected with a GFP-SHLD2 construct. 24 h post-transfection DNA damage was induced using Shield-1 and 4-OHT. The cells were then fixed and analyzed for the intensity of the GFP-SHLD2 signal at mCherry-LacR-Fok1 focus. Shown is the quantification of the GFP-SHLD2 signal at the Fok1 focus. Data are represented as a box-and-whisker plot where the whiskers represent the 10-90 percentile. At least 75 cells were counted per condition. 293T cells were transfected with Flag-REV7 and GFP-SHLD2 expression vectors as indicated. 24h post-transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU-60019 for 1 h prior to irradiation. 1h post-irradiation (10 Gy) nuclear extracts were prepared and REV7 complexes were immunoprecipitated using anti-Flag (M2) Resin and then analyzed by immunoblotting using GFP, REV7 and p-Chk1 antibodies. U2OS EJ5-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL). CH12F3-2 cells stably expressing the indicated shRNAs were stimulated with a cocktail of cytokines (CIT) to induce class switching to IgA. The percentage of IgA+ cells was monitored 24 and 48h post-stimulation by staining with an anti-IgA antibody followed by flow cytometry analysis. U2OS DR-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL). U2OS cells were transfected with the indicated siRNAs and then treated with CldU, IdU and NCS 48h post-transfection as indicated. The slides were stained, dehydrated, mounted and visualized and shown is the quantification of CldU/IdU tract length in order to visualize DNA end-resection (Right). At least 500 DNA tracks were measured per condition.
Figure 6(a) Kymograph showing the deformation of the cortex along a segment perpendicular to a laser cut (red line time point) in the E-YSL of a Myosin-GFP transgenic embryo [Tg (β-actin:myl 12.1-e-GFP)]. After a rapid opening, an active contraction displaces the cut centroid vegetalward indicating that the EVL is more easily stretched (elastic) than the vegetal yolk cellmembrane/cortex. When the gap is closed, the cut center returns to its initial position and the elastic equilibrium is restored. Scale bar 10 μm. (b) Proposed model of epiboly progression. The contractile E-YSL and the imbalance of stiffness between the EVL and the yolk surface account for epiboly progression. The vegetal boundary of the E-YSL is not mechanically free but linked to the tense yolk membrane. The tension originated by constriction of the actomyosin ring (E-YSL) is thus conveyed by the yolk membrane towards the vegetal pole leading to the progression of the less stiff EVL. Passive movements within the yolk arise as a result of force transmission from the surface. (c) Dynamic simulation of epiboly based on a single spherical cortex model (SC) considering a gradient of elastic tension from animal to vegetal and a constricting area at the E-YSL (from Movie EV8 - 40 %; 60 %; 80% epiboly). Color-coded mechanical power map normalized values in arbitrary units (positive - red, negative - blue) (left). Flow lines (right). The mechanical power is supplied at the surface and internal vortices and their shift from animal to vegetal are precisely simulated. (d) Dynamic simulation of AV and CC stresses normalized values in arbitrary units (from Movie EV9 - 40 %; 65 %; 80 % epiboly) represented as in Figure 1a. The AV and CC stresses are equal at the poles, as the epiboly simulation progresses, the CC stress steeps up from animal to vegetal and the difference between CC and AV stresses change sense at the EVL / yolk margin.
IEM detection of viral markers in MERS-CoV-infected cells.Fig. 6. IEM detection of viral markers in MERS-CoV-infected cells.(A-G) Immunogold labeling of thawed cryo-sections of MERS-CoV-infected Huh7 cells (12 hpi) for the detection of the indicated viral proteins. (A-C) Structural proteins were detected on virions (black arrowheads) and, for the M and S proteins, also on Golgi cisterna. While regions containing DMS (white arrowheads) and CM labelled for the N protein (D) and nsp3 (G), the M and S protein were not detected in these areas. (H-I) Immunogold labeling of dsRNA in HPF-FS samples of MERS-CoV-infected Huh7 cells (13 hpi). The label accumulated on DMVs, which could be easily detected in this type of samples (grey arrowheads), while the regions with CM and DMSs, which appeared as dark areas among the DMV clusters, where devoid of dsRNA signal. G, Golgi complex; m, mitochondria. Scale bars, 250 nm.
a) Ingenuity analysis of genes differentially expressed between KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes determined by RNAseq (set selection using DESeq with adjP0.f6(a) Ingenuity analysis of genes differentially expressed between KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes determined by RNAseq (set selection using DESeq with adjP0.05, fold-change>2 cutoffs). Biological functions overrepresented in the differentially expressed gene sets are shown with the respective Bonferroni-Hochberg P-value indicating enrichment significance. (b) Representative immunohistochemistry and quantitative assessment of γH2AX and phospho-Chk2 in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 18 weeks after AdCre inhalation. For γH2AX and phospho-Chk2 staining, sections were scored independently in a double-blinded manner. Data are shown as mean percentages (±s.e.m.) of positive cells per total cell numbers. n=6 mice per genotype. Scale bars, 50 μm. ***P0.001 (χ2-test of a generalized linear model with logit link assessing the genotype effect). (c) Representative histological lung sections of KRas;Atg5fl/+;p53fl/fl and KRas;Atg5fl/fl;p53fl/fl littermate mice16 weeks after AdCre inhalation. Scale bars, 2 mm. (d) Distribution of hyperplastic lesions, adenomas and adenocarcinomas (±s.e.m.) in KRas;Atg5fl/+;p53fl/fl and KRas;Atg5fl/fl;p53fl/fl littermates 16 weeks post AdCre infection (n=4 per genotype). (e) The Kaplan-Meier survival curves for KRas;Atg5fl/+;p53fl/fl (n=8) and KRas;Atg5fl/fl;p53fl/fl (n=9) littermate mice injected intranasally with AdCre (2.5 × 107 p.f.u.). There was no statistical difference between the cohorts (log-rank test).
Figure 6(A-B) Terminal cells expressing the membrane marker CD4::mIFP under btl-gal4 and FGFR::GFP under its own promoter (from the fTRG library). (A) Time lapse imaging of a control cell. Blue arrowheads point to filopodia and basal plasma membrane and red ones point to puncta containing CD4::mIFP and FGFR::GFP. (B) shibirets cell imaged before dynamin inactivation, after 23 and 45 minutes of inactivation, and after 30 minutes of recovery. White arrowheads point to FGFR::GFP accumulation at the apical compartment. (C-E) Single confocal planes of terminal cells expressing PH::mCherry and stained for βPS-Integrin. The outline of the cells was traced using the PH::mCherry signal and is shown as a blue dashed line. (C) Control. Blue arrowheads: βPS-Integrin signal in filopodia. (D) 1 hour at 34ºC, (E) 2 hours at 34ºC. (C''-E'') Orthogonal views of the lines shown in white in (C-E). (F-G) Quantification of fluorescence intensity from stained embryos of FGFR::GFP (F) and of βPS-Integrin (G). Data are plotted as mean +/- SD; significance was assessed using one-way ANOVA with Dunnett's correction for multiple comparisons. **p=0.0022, ***p=0.0003, ****p<0.0001. Number of cells analyzed, for (F): Control n=8, 1hr at 34ºC n=6, Recovery n=8. (G). For (G): Control= 7, 1hr at 34ºC= 8, 2hrs at 34ºC= 8.
Figure 5Cideb interacts with Sec12. 293T cells transfected with Cideb and GFP-tagged Sec12, Sec23, Sec16S, or Sec16L were subjected to immuno-precipitation with an anti-Cideb antibody, and levels of the co-immunoprecipitated protein were detected by an anti-GFP antibody. Co-localization of SCAP and Cideb at the ER exit sites. HepG2 cells transfected with GFP-SCAP and HA-Cideb were arrested with 10 µM BFA and 1 µM Nocodazole for 2 hours, then fixed with methanol and stained with antibodies against Sec12 or HA. 3D reconstruction was obtained by processing 3D-original image with Imaris. Scale bars represent 10 µm (Merge), and 1 µm (Inlay and 3D reconstruction). Localization of Cideb at the ER exit sites. AML12 cells knocked-in with a GFP tag at the C-terminus of Cideb genome was treated with 10 µM BFA for 1 hour, then fixed with methanol and stained with antibodies against Sec12. Scale bars represent 10 µm (Merge), and 1 µm (Inlay). Sec12 interacts with the 118-165 portion of Cideb. HA-tagged Sec12 was co-expressed with the indicated Flag-tagged Cideb truncation mutants in 293T cells and immuno-precipitated with an anti-Flag antibody. Levels of the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE. The schematic diagram of Cideb and its truncations. Sec12/Cideb interaction modelled by molecular dynamic simulation. The red portion highlighted the predicted domain of Cideb interacting with Sec12. Lysine 128 of Cideb is required for the interaction with Sec12. 293T cells transfected with HA-Sec12 and the indicated Flag-tagged Cideb constructs were subjected to immunoprecipitation with an anti-Flag antibody, and levels of the co-immunoprecipitated HA-Sec12 were detected with an anti-HA antibody. Recombinant Cideb interacts with Sec12. GST-Sec12 and MBP-tagged Cideb or Cideb-K128A were purified from E.coli and subjected to in vitro binding. Cideb-K128A fails to promote SREBP processing. Cideb-/- mice were injected with adenovirus expressing GFP, Flag-Cideb, or Flag-Cideb-K128A, and sacrificed 7 days after injection. Liver lysates were subjected to IB with the indicated antibodies following SDS-PAGE and quantification of IB data from 3 independent experiments. Data represent Mean ± SEM; **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test. Cideb, but not Cideb-K128A, promotes SCAP/SREBP incorporation into COPII vesicles. Liver microsomes were isolated from mice expressing the indicated proteins and subjected to in vitro budding assay. Resulting vesicles and the donor microsomes were subject to IB with the indicated antibodies following SDS-PAGE.
Figure 4A) NK cells were stimulated with either IL-2 or IL-15 in the presence or absence of PGE2 for two days and thereafter purified as CD25+/CD54+ and CD25-/CD54- and tested in a 51Cr-release assay against K562. (E: T=5:1, n=4, biological replicates). Stars indicate significant p-values comparing CD25+/CD54+ vs. CD25-/CD54- NK cells for each condition. B-F) NK cells were stimulated with IL-15 and purified as CD25+/CD54+ and CD25-/CD54- and co-cultures with K562 cells (5:1 ratio) for six hours and analyzed for the expression of B) CD107a, C) IFNγ, D) Perforin, E) TRAIL and F) Granzyme B were by flow cytometry (n=4, biological replicates). G) Flow cytometry analysis of levels (Left=% frequency, right=fold change of mean fluorescence intensity) of phosphorylated S6 (S235/236) in CD25+/CD54+ and CD25-/CD54- IL-15 activated NK cells (n=4, biological replicates). H) Flow cytometry analysis of PDE4A expression in CD25+/CD54+ and CD25-/CD54- IL-15 activated NK cells (n=4, biological replicates). I) Representative image of PDE4A expression in CD25+/CD54+ and CD25-/CD54- IL-15 activated NK cells. Scale bar: 10 µm. J) NK cells were first stimulated with IL-15 for two days in the absence of PGE2, then purified as CD25+/CD54+ or CD25-/CD54- NK cells and tested in a 51Cr-release cytotoxicity assay against K562 cells at an E:T ratio of 5:1 in the presence (filled bars) or absence (open bars) of PGE2 (1 µM) (n=3, biological replicates). K) Mitochondria mass from purified CD25+/CD54+ and CD25-/CD54- NK cells measured by flow cytometry (n=5, biological replicates). L) Representative image of Mitotracker Red FM in CD25+/CD54+ NK cells and CD25-/CD54- NK cells. Scale bar: 20 μm.
Oxidant accumulation in wild type and atg mutant plants.Figure 6(A) NBT treated Arabidopsis (Col-0 and two independent atg8a mutant lines) following agar plug inoculation with the A2 mutant. Images were collected 48 hours post inoculation. Dotted lines represent the edge of the observable legion. (B) RT-PCR was used to evaluate the transcript levels of three catalases (CAT1, 2, and 3) and three superoxide dismutases in Col-0 plants following inoculation with wild type S. sclerotiorum (black bars) and the A2 mutant strain (grey bars). *>2 fold change, **>5 fold change.
Coordinated regulation of ATHB2 and FT by PIF4.Figure 6Schematic of the regulation of ATHB2 and FT expression by PIF4 and temperature. Comparison of data and model simulations for temperature response of PIF4 (B, C) and ATHB2 (D, E) mRNA in LDs (16L:8D) in the WT. Data from Nomoto et al (2012a) were normalised such that data and simulation have equal peaks at 28°C (22°C data: black lines, filled squares; 22°C simulation: black lines; 28°C data: red lines, filled squares; 28°C simulation: red lines). CO mRNA data in LDs in WT and the pif4;pif5 mutant at 22 and 27°C (WT 22°C data/simulation: as in (B-E); pif4;pif5 22°C data: dashed black lines, open squares; WT 27°C data: red lines, filled circles; pif4;pif5 27°C data: dashed red lines, open circles). CO protein data in LDs (16L:8D) in WT at 22 and 27°C (as in F). FT mRNA data in LDs in WT and the pif4;pif5 mutant at 22 and 27°C (as in F) FT mRNA simulation in LDs in WT and the pif4;pif5 mutant at 22 and 27°C (WT 22°C simulation: as in B-E; pif4;pif5 22°C simulation: dashed black lines; WT 27°C simulation: red lines; pif4;pif5 27°C simulation: dashed red lines).