EMBO/sd-character-level-ner huggingface dataset:
Figure 2a, Representative flow-cytometry plots of CD44 and CD62L expression on total CD8 T cells of 45-wk-old Dj-1 KO and age- and sex-matched WT mice. b-c, Percentages of CD44low CD62Lhigh (Tn) (b) and CD44high CD62Llow (Tem) (c) cells among total CD8 T cells of spleen and pLNs from young and 45-wk-old Dj-1 KO and WT littermates (young KO, n=5; young WT, n=5; 45-wk-old KO, n=8; 45-wk-old WT, n=6; for 45-wk-old mice, data pooled from 2 independent experiments). d, Representative histogram overlay of PD-1 expression among total CD8 T cells in spleen of 45-wk-old mice (left panel) and percentages of PD-1+ cells among total CD8 T cells (right panel). e, Percentages of Ki-67+ cells among total CD8 T cells. f, Percentage of Ki-67+ cells among splenic CD8 Tn, Tem and Tcm in 45-wk-old Dj-1 KO and age- and sex-matched WT mice. g g, Representative flow-cytometry plots of Ki-67 and PD-1 among splenic CD8 Tem of 45-wk-old mice. h, IFN-γ production in CD8 T cells of spleen and pLNs after in vitro stimulation using 50 ng/ml of PMA and 750 ng/ml of ionomycin for 5 h. i, Represenative flow-cytometry plots of CD44 and CD49d among blood CD8 T cells of 60-wk-old mice. j, Percentages of Tn (top), Tvm (middle), Tmem (bottom) among 60-wk-old or young mice (young KO, n=4; young WT, n=3; 60-wk-old KO, n=4; 60-wk-old WT, n=5). k, Represenative histogram of CD31 among blood CD8 T cells of 60-wk-old mice. l, CD31 MFI of total CD8 T cells (young KO, n=4; young WT, n=3; 60-wk-old KO, n=4; 60-wk-old WT, n=5).
Figure 2Investigating the effect of sustained oxidative mitochondrial metabolism on the transition from naïve ESCs to primed EpiLCs through supplementation of the glycolysis inhibitors 2-deoxy-D-glucose (2-DG) and dichloroacetate (DCA), respectively. Flow cytometry analysis of Rex1-GFPd2 cells following addition of 10mM 2-DG during the 48h EpiLC induction. Representative GFP intensity distributions are depicted. Average proportions of Rex1-GFPd2 positive (GFP+) cells are quantified from two independent biological replicates. Expression analysis by qRT-PCR of naïve pluripotency and epiblast marker genes in bulk 48h cells after 10mM 2-DG treatment. Relative expression levels, normalized to control culture conditions, are shown. Graphs represent averages from triplicate (duplicate for Klf4 and Tfcp2l1) independent biological experiments. Colony-forming ability following 10mM 2-DG supplementation during EpiLC stimulation. Representative images of alkaline phosphatase (AP)-stained colonies are displayed. Scale bar, 250 um. The average colony formation, normalized to control culture conditions, quantified from two independent biological replicates, is shown.
Figure 10(A) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63(ycbB) was performed with the pHV30 vector or recombinant plasmids encoding mepA, mepK, mepS or derivatives encoding catalytically inactive endopeptidases under the control of the PrhaBAD promoter. (B) The complementation assay was repeated for BW25113 Δ7EDs pHV63(ycbB), which was obtained by deletion of all chromosomal endopeptidase genes except mepM. Induction of ycbB and of endopeptidase (ED) genes was performed with 0.2% L-arabinose and 1% L-rhamnose, respectively. BHI agar plates contained chloramphenicol (20 µg/ml) to counter-select loss of pHV63(ycbB).
The eIF4F complex regulates TORC1.Figure 3(a) Knockdown of eIF4F components (eIF-4E and eIF-4G) partially phenocopy the eIF4A knockdown, leading to elevated TORC1 activity upon amino acid removal (30 min). Knockdown of all other tested translation initiation factors do not cause elevated TORC1 upon amino acid removal (main Figures 1c, 2a' and 2b). Error bars: std. dev. n=3 biological replicates. (b) Quantification of de novo protein synthesis rates by OPP incorporation reveals that eIF4E and eIF4G knockdowns deplete eIF4F function less efficiently than the eIF4A knockdown, explaining why the effects of eIF4E and eIF4G knockdowns on TORC1 activity (panel a) are a bit milder than the eIF4A knockdown. Kc167 cells treated with dsRNA for 4 days then incubated with 20μM Click-it OPP reagent 30min before fixation and fluorescent labeling. Quantification of OPP fluorescence per cell (nuclear count) for two independent experiments is displayed (3 independent images per condition), normalized to the no dsRNA condition. Scale bars: 25µm.
Figure 3A) Q-PCR analysis of TEAD1-4 mRNA expression in control versus Lats1/2 dKO intestines at 7-days post i.p. injection with tamoxifen. Note the strong increase in TEAD1 and TEAD4 mRNA levels in particular, which indicates that these are both target genes of YAP signalling. B) RNAscope in situ hybridisation analysis of TEAD1/2/4 mRNA expression from control (Cre negative) Lats1flox/flox Lats2flox/flox animals and Villin-CreERt Lats1flox/flox Lats2flox/flox animals treated with tamoxifen to induce homozygous deletion of Lats1/2 (dKO). Tamoxifen treated (3 days i.p.) Villin-CreERt Lats1flox/flox Lats2flox/flox double homozygous mouse intestines show elevated expression of TEAD1 and TEAD4 in both the small intestine (top) and colon (bottom). Tissues were harvested and analyzed at day 7 after first tamoxifen injection. (n=5 animals for each genotype) C) Q-PCR analysis reveals a progressive increase in the YAP-TEAD target gene Ctgf in Lats1/2 dKO intestines at 3-days and 7-days post i.p. injection with tamoxifen, as well as similarly increased TEAD1 and TEAD4 expression, confirming that TEAD1 and TEAD4 are YAP-responsive genes. Notably, Axin2 and TEAD2 exhibit a mild but progressive decline over 3 to 7 days after Lats1/2 deletion, consistent with Axin2 being a known Wnt-specific target gene, and with Wnt signalling remaining active in Lats1/2 dKO intestines.
Figure 3A and B. Initial colonization of gnotobiotic (A) or SPF (B) mice (n=4 mice per group) with N1 followed by secondary challenge with N1 (closed squares) or E1 (open squares). FecalCFU was determined for the primary and secondary colonization strains through four weeks post-secondary challenges. C. All primary colonization and secondary challenge pairs were tested with 3 NTBF and 2 ETBF strains. Stable colonization of the secondary challenge strain significantly above the limit of detection is denoted as a '+' while non-significance is denoted as a '−' (n=4 mice per group). The diagonal gray bar indicates self secondary challenge, the horizontal red bars show strains that provide broad colonization resistance against non-self strains and the vertical dashed box indicates a strain that has an enhanced secondary colonization phenotype.
Figure 3A, B The oocytes were exposed to different concentrations of NO3- in N free MBS for 1.5 h at pH5.5. Net NO3- accumulation was fitted with both Michaelis-Menten and Linear Regression in A for high affinity range and Linear Regression in B for low affinity. Values are the mean ± SE (biological replicates: n=3) with 3-4 oocytes in each sample.
Figure 8(A and B) XO8 GSCs were subcutaneously (s.c.) injected in the flank of mice. When the tumor volume reached approximately 180 mm3, GSC-bearing mice were administered intraperitoneally (i.p.) with 1 mg/kg MG132 and 10 mg/kg STK alone or in combination on every day for 2 weeks. Tumors were dissected from the subcutaneous regions of nude mice. Inset picture shows the tumor in respective group. Therapeutic effects were monitored using bioluminescence imaging. (C) The bar diagram represents the tumor volumes. (D) Tumor weights were determined at the end of the study. (E) The tumor sizes in each group were determined by histological analysis at day 21 after tumor inoculation. (F) XO10 GSC were orthotopically injected into the brains of BALB/c nude mice. STK047915, MG132, or both were injected intraperitoneally to GSCs-bearing mice at day 14 after XO10 (5×105 cells) inoculation. The PBS was used as a control. Representative photographs of H&amp;amp;E staining from each group show tumor growth. Magnification, ×1.
Figure 4A-D. Immunofluorescence images with Proteostat (red) and DAPI (blue) staining on (A) human normal skin, (B) samples of primitive melanomas, (C) melanoma metastases in brain and (D) melanoma metastases in lung. E. Details of brain metastases. Scale Bar is 30µm. F. Quantitation of Proteostat positive dots in primitive vs metastatic melanoma tissues: 6 tissues from metastatic lesions and 6 from primitive melanoma lesions were analysed. For each tissue two sections were quantified. T-test analysis was applied. T-test analysis, ** = p-value<0.01 (N=6, data are mean ±SEM).
Epiblast cells organize as rosettes at the onset of primitive streak morphogenesis.Figure 1(a) Manually segmented rosettes on 3D reconstructions from Z-stacks of 25 μm of an E5.75 embryo imaged using lightsheet microscopy. AVE cells are in green, and all cell membranes are labelled in grey. Left: anterior view; Middle: lateral view (anterior to the left); Right: posterior view. Scale bars: 20 μm. (a') Mouse line strategy used to perform live imaging. (a") Left: Number of rosettes normalized by the total number of cells in the epiblast region in focus expressed in percentage. Right: Frequency of rosettes, normalized by the area in mm2 of the epiblast region in focus and the time of observation in minutes. Data information: Mean ± SEM, n=41 frames from 3 embryos. (b, c) Rosettes outlines on optical slices from E5.75 (b) and E6.25 (c) embryos imaged by confocal microscopy. AVE cells are in green, and all cell membranes are labelled in grey. Left: anterior view; Middle: lateral view (anterior to the left); Right: posterior view. Scale bars: 20 μm. (b') Mouse lines strategy used to perform live imaging. (b'', c') Left: Number of rosettes normalized by the total number of cells expressed in percentage. Right: Density of rosettes, normalized by the area in mm2 of the epiblast region in focus. The posterior region includes the PS region, as it could not be precisely discriminated. Data information: Mean ± SEM. E5.75: n anterior=19 frames from 3 embryos, n lateral=23 frames from 5 embryos, n posterior=20 frames from 3 embryos. E6.25: n anterior=16 frames from 2 embryos, n lateral=12 frames from 1 embryo and n posterior=16 frames from 2 embryos.
Figure 1A. Confocal microscopy analysis showing the localization of endogenous Rab22a (green) and different endocytic markers (red): early endosomal marker EEA1, late endosome/lysosomal marker Lamp1 and the recycling compartment marker TfR in BMDCs at steady state. The means ± SEM of the Pearson correlation coefficients (Pcc) from Rab22a and EEA1 (0.4317 ± 0.05101), Lamp1 (0.8412 ± 0.03562) and TfR (0.6409 ± 0.02999) were estimated from 15 images analyzed for each marker. Scale bars: 5 µm. B. Colocalization of endogenous Rab22a (red) and EEA1 (magenta) around endosomes containing fluorescent soluble OVA (OVA-FITC, green) after 30 min of internalization by BMDCs. More than 60% of the endosomes were positive for Rab22a and EEA1. The indicated boxes are shown at higher magnification in the insets. Scale bars: 5 µm. C. IF detection of endogenous Rab22a (green) and Lamp1 (red) after 1 h of phagocytosis of 3 µm latex beads (LB) in BMDCs. Around 50% of the phagosomes were double positive for Rab22a and Lamp1. Asterisks indicate the LB. Scale bars: 5 µm. D. BMDCs were incubated with 3 μm magnetic beads for 15 min at 37°C and chased for 0 or 45 min. The panel shows immunoblotting of purified phagosomes and the total cell lysates (TCL) analyzed for Lamp1 and Rab22a. A total protein amount of 10 µg and 50 µg was loaded for purified phagosomes and TCL, respectively. The blot is representative of three independent experiments.
Figure 7 The effect of kinase inhibitors on EDN induced cell migration of UACC257 cells was analysed using a scratch assay. Scratches were made in UACC257 monolayers. Serum was removed and cells were treated with PBS or 100 nM EDN in combination with DMSO or kinase inhibitors at 0.1 μM or 1 μM in triplicates. A Example pictures showing the effect of EDN on migration. Cell free areas are delimited by blue lines. Migration was quantified as the change in cell free area between 0 h and 48 h. B The four inhibitors that were effective at 1 µM are shown in detail, i.e. every point corresponds to one well. C Abundance of E-Cadherin (normalized to GAPDH and Actin) was assessed by LC-SRM after 48 h treatment with EDN or kinase inhibitors at 1 µM. Only the single inhibitor which could reverse EDN induced E-Cadherin repression is shown. * p < 0.05 two-sided unpaired t-test.
Figure 5Comparison of changes in gene expression induced by Bhlhe40/Bhlhe41-deficiency in steady-state knockout mice (horizontal axis) and in mixed BM chimeras (vertical axis). Log2 transformed DKO/WT fold changes are plotted. Genes with expression of > 5 TPM in at least one of the samples are displayed. Genes highlighted in magenta indicate known and putative regulators of AMs. Closed symbols - Bhlhe40-bound genes (Bhlhe40 ChIP-seq peak assigned to the nearest gene within a 50 kb window). Open symbols - genes without Bhlhe40 binding. Presence of RNA transcripts (RNA-seq; top) at the Gas7 and Acaa1b genes in WT and DKO AMs; the ChIP-seq analysis of H3K27 acetylation in WT and DKO AMs (middle) and Bhlhe40 binding in WT AMs (bottom). All analyses performed on ex vivo AMs. Consensus Bhlhe40-binding motif identified by de novo motif-discovery (MEME-ChIP suite; E-value, 8.6 × 10−121). Distribution of Bhlhe40 peaks at intergenic regions, the gene body or the promoter in AMs. Gene set enrichment analysis (GSEA) on dataset ranked by DKO/WT fold change in steady-state AM RNA-seq using set of genes assigned to top 1,000 Bhlhe40 peaks (ranked by P value) (left) or a random set of 1,000 genes (right). Similar results were obtained with all three random sets tested. NES - normalized enrichment score. FDR False Discovery Rate. Pairwise comparison of H3K27 acetylation in WT and DKO AMs performed on 2-kb regions around the Bhlhe40 peak summits (right - for all Bhlhe40 peaks called with P value of < 10−10; center - for top 3,000 Bhlhe40 peaks (ranked by P value)) and around WT AM ATAC-seq peaks (Immgen) that did not have a Bhlhe40 peak in their close proximity (filtered as described in Materials and Methods) (left). Volcano plots show adjusted P values (vertical axes) and log2 fold change (horizontal axes) for H3K27ac pairwise comparison in WT vs DKO AMs; genes assigned to some of the Bhlhe40-binding regions are labeled on the left plot.
Figure 5 Cultured hippocampal neurons were infected at 14 DIV with lentivirus for expression of both shRNA against PSD-95 (sh95) and sh-resistant PSD-95-EGFP. Thus endogenous PSD-95 was replaced with either wild-type (WT), E17R, or T19K PSD-95. Cultures were treated with or without BIC (50 μM) at 17 DIV for 24-48 h or left untreated before fixation and staining for surface GluA1. (A) Representative confocal microscopic images of PSD-95-EGFP fluorescence (green) and surface GluA1 immunostaining (red) at low (top panels; scale bar: 20 µm) and high enlargement (bottom panels; scale bar: 5 µm). (B) Quantification of surface GluA1 intensity from (A) showed reduction in AMPAR surface levels during scaling down in neurons expressing WT PSD-95 but not in those expressing either E17R or T19K mutant. Three dendritic segments from each of 31-42 neurons from three independent sets of cultures were analyzed per condition (**p<0.01; n.s., not significant; one-way ANOVA followed by Bonferroni's post hoc test).
Figure 1(A) Schematic diagram of the Pax5Jak2 allele. Human cDNA sequences, starting in PAX5 exon 4 and encoding the remaining PAX5-JAK2 protein, were inserted in frame into exon 4 of the mouse Pax5 locus to generate the Pax5Jak2 allele The C-terminal tag sequence (grey) contains an epitope for the V5 antibody, two cleavage sites for the TEV protease and a biotin acceptor sequence (Biotin). A black oval denotes the B cell-specific enhancer (En) in intron 5 pA, polyadenylation site. Notably, the human and mouse Pax5 protein sequences encoded from exon 1 to exon 5 contain only one amino acid substitution (human Ser13 vs mouse Ile13 in exon 1) located upstream of the paired domain (B) Expression of the Pax5 protein in Pax5Jak2/+ pro-B cells. Nuclear extracts of short-term cultured pro-B cells from 3-week-old Pax5Jak2/+ and Pax5+/+ mice were analyzed by immunoblot analysis with an anti-Pax5 antibody recognizing the N-terminal paired domain. The size of marker proteins is indicated in kilodaltons (kDa) to the left. (C) Flow-cytometric analysis of bone marrow cells from the two hindlegs of 3-week-old Pax5+/+ and Pax5Jak2/+ mice. The percentage of cells in each gate or quadrant is indicated. (D) Absolute cell numbers of the indicated cell types were determined by flow-cytometric analysis of the bone marrow from 3-week-old Pax5+/+ and Pax5Jak2/+ mice. Average cell numbers are shown with SEM and were statistically analyzed by multiple t-tests (unpaired two-tailed with Holm-Šídák's correction); ns, not significant (P > 0.05). One of 3 independent experiments is shown. (E) Flow-cytometric analysis of bone marrow cells and splenocytes from Pax5Jak2/+ mice at the age of 4 or 6 weeks. The newly emerging B220low B cells are highlighted by black boxes. (F) Kaplan-Meier survival analysis of Pax5Jak2/+ (black) and control Pax5+/+ (grey) mice. n, number of mice analyzed. A P value of < 0.0001 was determined for the survival curves by statistical analysis with the log-rank (Mantel-Cox) test. (G) Size comparison of the spleen and lymph nodes from a control Pax5+/+ mouse and a Pax5Jak2/+ tumor mouse. (H) Eosin-hematoxylin-stained sections of the lung and liver of a Pax5Jak2/+ tumor mouse. Infiltrating and blasting tumor cells are indicated by an arrow. (I) Flow-cytometric analysis of lymph node cells from a control Pax5+/+ mouse and a 10-week-old Pax5Jak2/+ tumor mouse. (J) Flow-cytometric analysis of B220lowCD19+ B cells from the bone marrow of a 4-week-old Pax5Jak2/+ mouse.
Figure 3(A) Raji-Env cells were subcloned to obtain cell lines expressing increasing amounts of Env. Env surface levels were determined by the binding of 10-1074 and measured by flow cytometry. One representative experiment is shown. (B) Parental Raji cells and the different Raji-Env clones were cultured with 10-1074 or an isotype control (mGO53) and NHS for 24h. The % of CDC was calculated as the relative percentage of dead cells compared to the "no antibody" condition. n=3 donors of serum. (C) Non-infected (NI) and HIV-1-infected primary CD4 T cells (strain NLAD8) as well as three Raji-Env clones (D1, D2 and D4) were incubated with 10-1074 and antibody surface levels were analyzed by flow cytometry. Results are expressed as median fluorescence (MFI) of staining in total cells (Raji, n=3 independent experiments) or in infected (Gag+) cells (CD4 T cells, n=5 donors). (D) HIV- or HIV∆Vpu-infected CD4 T cells (strain NLAD8) were subjected to antibody binding with indicated antibodies and surface levels were determined by flow cytometry (left panel). The staining obtained with mGO53 was similar to the background signal observed on unstained cells. Infected cells were cultured with the indicated antibodies and NHS for 24h and the C3 surface levels on infected (Gag+) cells were determined by flow cytometry (right panel). One representative experiment is shown. (E) HIV- or HIV∆Vpu-infected CD4 T cells (strain NLAD8) were incubated with NHS and either 10-1074 (left panel) or 3BNC117 (right panel). Both WT or E430G-mutated antibodies were used. After 24h, C3 surface levels on infected cells were analyzed by flow cytometry. Results are expressed as MFI of staining on infected (Gag+) cells. The MFI of C3 staining on Gag- cells was included as a control. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.
Figure 4TGFB1 and ITGA5 loci on chromosome 19q13.2 and chromosome 12q13.13, respectively. The location of putative CTCF binding sites and representative amplicon sites used for qPCR are shown with names below. A ChIP assay was performed for RAD21KD-MCF7 or -SNU16 cells using antibodies specific for (A) RAD21, (B) Pol II, (C) AcH3, or IgG (as a control). Enrichment was measured by qPCR and expressed relative to the total input (4%). ChIP signals for AcH3 were normalized to total H3. Results for at least three chromatin preparations are shown ± S.E.M. *P < 0.01, **P < 0.05. Statistical significance was validated by Student's t-tests. Relative crosslinking frequencies among CTCF/RAD21 binding sites on the genes were measured with a 3C assay in the control GFP-shRNA (blue line) or RAD21KD (red line) -MCF7 or -SNU16 cells on day 30 after lentiviral transduction. (D) BamH1 or (E) Xba I restriction sites on the TGFB1 or ITGA5 gene, respectively, appear as gray shaded bars. Black shading indicates the anchor fragment. Each value was normalized to crosslinking frequency at the ERCC3 gene. The maximum crosslinking frequency was set at 1. (means ± S.E.M, n = 3). *P < 0.01, **P < 0.05. Statistical significance was validated by Student's t-tests.
Figure 1(A) Bacterially purified 6xHis-rRv2626c and analyzed by Coomassie blue staining (left) or immuno blotting (IB) with αHis (right). (B) BMDMs were incubated with rRv2626c for the indicated times and then cell viability measured with MTT assay. (C) Fluorescence confocal images showing in BMDMs treated with Rv2626c (2.5 μg/ml) for 30 min, fixed, immunostained with antibodies for His (Alexa 488) and DAPI. Scale bar, 10 μm. (D and E) BMDMs from WT mice were treated with LPS (100 ng/ml) or rRv2626c for indicated times (D). BMDMs from WT, TLR2-/-, TLR4-/-, MyD88-/-, TRIF-/-, IRAK1-/-, TRAF6-/-, TBK1-/- mice were treated with 2.5 μg/ml rRv2626c or rVector for 18 h (E). Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA. (F) BMDMs were treated with 2.5 μg/ml rRv2626c for 1 h, harvested and then subjected to IB for αIκBα and His. αActin was used as a loading control.
SARS-CoV-2 miR-O7a.Figure 4(A) Base-pairing of CoV2-miR-O7a.2 to complementary 3´UTR site or site with mutations in seed region cloned in 3'UTR of firefly luciferase mRNA in pmirGLO Dual-Luciferase miRNA Target Expression Vector. The seed region required for binding of miRNAs with its target is underlined. (B) Relative firefly luciferase normalized to Renilla luciferase of pmirGLO Dual-Luciferase miRNA Target Expression Vector with either CoV2-miR-O7a.2 target site or CoV2-miR-O7a.2 site with mutations in seed region at 24 h on co-transfection with CoV2-miR-O7a.2 or control miRNA mimics. Activity on transfecting control miRNA mimic was considered 100 %. Bars and error bars represent the average and standard deviation from three independent experiments. (C) Base-pairing of CoV2-miR-O7a.2 to complementary 3´UTR site of BATF2 mRNA. The seed region required for binding of miRNAs with its target is underlined. One mismatch in the seed region of CoV2-miR-O7a.1 is observed. (D) Kinetics of BATF2 and LAMP3 mRNA levels in non-induced and interferon IFN-α-induced (for 8, 16, and 24 h) A549-ACE2 cells transfected with either control or CoV2-miR-O7a.2 mimics. Normalized read abundances in transcript per million (TPM) are shown for two individual experiments (n=2). (E) Genomic view of the human BATF2 gene showing normalized RNA-seq reads from non-induced and IFN-α-induced (for 8 h) A549-ACE2 cells transfected with either control, CoV2-miR-O7a.1 or CoV2-miR-O7a.2 mimics. The complementary site of CoV2-miR-O7a to BATF2 3´UTR is shown.