EMBO/sd-character-level-ner huggingface dataset:
Figure 1Percentage of circulating highly differentiated CD4 T cells within CD4 cells (upper graph) in age-matched healthy donors or NSCLC patients before undergoing immunotherapies. G1 and G2, groups of patients classified according to high THD cells (G1, >40% CD4 THD cells) and low THD cells (G2, <40% CD4 THD cells). Relevant statistical comparisons are shown by the test of Mann-Whitney. In green, objective responders (OR). In red, no OR. Below the graph, correlation of objective responses to G1 and G2 groups by the Fisher´s exact test. Waterfall plot of change in lesion size in patients with measurable disease classified as having a G1 (blue) or G2 (red) profile. Dotted lines represents the limit to define significant progression (upper line) or significant regression (lower line). ROC analysis of baseline CD4 THD quantification as a function of objective clinical responses. Kaplan-Meier plot for PFS in patients treated with immunotherapies stratified only by G1 (green) and G2 (red) CD4 T cell profiles. Patients starting therapy with a G2 profile had an overall response rate (ORR) of 0% and 82% of them experienced progression or death by week 9. ORR was 44.8% for G1 patients, and the 12-week PFS was 50.2%.
Figure 1(A) Overview of the tissues and organs analyzed based on transcriptomics by the three independent consortia Human Protein Atlas (HPA), FANTOM and GTEx. In total, 16 organ systems (with several tissues comprising an organ system) were used to create a consensus normalized expression (defined as the unit NX) based on the expression levels of all three datasets. (B) ACE2 gene expression summary in human tissues in 61 different tissues and cells in NX. Cut-off for what is regarded as expressed was set to 1.0 NX.
Figure 1A TRAF6 expression in differentiating CD4+ T cells. Naïve CD4+ T cells were obtained from wild type C57BL/6 mice by FACS and activated with anti-CD3/CD28 (1µg, and 2µg/ml) for the indicated times in the presence of distinct Thelper lineage-directing cytokines or under neutral activation conditions (Th0). After total RNA extraction and cDNA conversion, RT-PCR determined of Traf6 mRNA in differentiating Th0, Th1, Th17 and iTregs was determined. B TRAF6 mRNA expression by human Tregs and non-Treg CD4+ T cell. Human Tregs (CD3+/CD4+/CD8-/CD25HIGH/CD127low/CD39+) and non-Treg CD4+ T cells (CD3+/CD4+/CD8-/CD25-) were obtained from the peripheral blood of healthy donors by FACS after Ficoll-Paque PLUS gradient centrifugation and magnetic bead enrichment of CD4+ T cells. TRAF6 mRNA was measured by qRT-PCR. C, D Evidence of lymphoproliferative disease in Traf6fl/flFoxp3Cre+ mice. (C) Spleens and lymph nodes were recovered from Traf6fl/flFoxp3Cre+ mice and Traf6fl/fl littermates at 8 weeks of age (Scale bars: 5 mm). (D) The cellularity of the lymphoid tissues of Traf6fl/flFoxp3Cre+ mice and their Traf6fl/fl Foxp3Cre- littermates was determined (8 mice/group). E, H Effect of Treg-specific TRAF6-deficiency on baseline T cell activation. The frequencies of effector cells (CD44high/CD62Llow), memory cells (CD44high/CD62Lhigh) and naïve cells (CD44low/CD62Lhigh) in the CD4+ T cell compartments of Traf6fl/fl and Traf6fl/flFoxp3Cre+ mice were determined by flow cytometry (5 mice/group). F, I Effect of Treg-specific TRAF6-deficiency on baseline T cell activation. The frequencies of effector cells (CD44high/CD62Llow), memory cells (CD44high/CD62Lhigh) and naïve cells (CD44low/CD62Lhigh) in the CD8+ T cell compartments of Traf6fl/fl Foxp3Cre- (wild type) and Traf6fl/flFoxp3Cre+ mice were determined by flow cytometry (5 mice/group). G, J Impact of TRAF6-expression on in vitro Treg differentiation. naïve CD4+ T cells were isolated from Traf6fl/flFoxp3Cre+ and Traf6fl/fl mice and differentiated into iTregs. Conditions of sub-optimal TGFβ concentrations (0.5ng/ml, 0.05ng/ml) were tested as well and intracellular FOXP3 was measured after 4 days.
Figure 5A In vitro pull-down assay showing the interaction of NMT1 and EGR2. Recombinant GST-NMT1 was incubated with immunoprecipated MBP-His-EGR2 or MBP-His at 4°C for 2 h. Anti-GST and anti-MBP were used to detect NMT1, EGR2 and MBP-His, respectively. Co-localization of NMT1 and EGR2 in N. benthamiana leaves. Scale bar: 50 μm C, D Effect of cold stress on the interaction of EGR2 and NMT1 determined by co-IP assay. Arabidopsis protoplasts expressing HF/NMT1-Myc and HF-EGR2/NMT1-Myc were treated with or without 4°C for 2 h. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-Myc and anti-HA were used to detect NMT1-Myc and HF-EGR2, respectively. In (C), relative band intensity is shown below the co-IP blot. In (D), each bar represents the mean ± SEM of three biological experiments (**P < 0.01, two-tailed t-test). E In vitro pull-down assay showing decreased interaction intensity of EGR2 (4°C) and NMT1 compared with that of EGR2 (22°C) and NMT1. Total proteins prepared from two-week-old EGR2-Myc plants grown at 4°C for 0 h and 6 h were immunoprecipated by anti-Myc agarose beads and incubated with GST-NMT1 in pull down buffer for 2 h at 4°C. Anti-Myc and anti-GST antibodies were used to detect EGR2 and NMT1 proteins. Relative band intensity is shown below the output blot. F Expression of NMT1 in NMT1-Myc overexpression plants. EF1α was used as a loading control. Freezing phenotype (G) of NMT1-Myc overexpression transgenic plants. Two-week-old Super:NMT1-Myc plants were treated at −5°C (NA) or −8°C for 0.5 h (CA). survival rate (H) of NMT1-Myc overexpression transgenic plants. Two-week-old Super:NMT1-Myc plants were treated at −5°C (NA) or −8°C for 0.5 h (CA). In (H), each bar represents the mean ± SEM of three biological experiments, and asterisks indicate significant differences compared with the wild type for the same treatment (*P < 0.05, two tailed t-test). OST1 kinase activity in NMT1-Myc overexpression plants under cold stress. Top, autoradiograph; bottom, CBB staining. Representative pictures are shown in (I) In (I): the ratio of band intensity of OST1 to RuBisCO in wild type with cold treatment for 2 h was set to 1.00. OST1 kinase activity in NMT1-Myc overexpression plants under cold stress. the relative kinase activity is shown in (J). In (J): each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two tailed t-test).
Figure 2A) Three-dimensional digital reconstructions of optical sections through immunofluorescently stained mesenteric resistance arteries from wild type, Tek-Cre::Lama5-/- and Lama4-/- mice, and optical sections through vessel walls to show the endothelial and underlying smooth muscle layers. DAPI staining permits identification of endothelial cell nuclei, which lie perpendicular to the nuclei of the smooth muscle cells. Laminin α5 is absent from endothelial basement membranes of Tek-Cre::Lama5-/- arteries (arrow) but is still present in smooth muscle basement membranes (asterisk), while laminin α4 is still detectable in both endothelial and smooth muscle basement membranes. Lama4-/- arteries lack laminin α4 in both endothelial (arrow) and smooth muscle (asterisk) basement membranes, but laminin α5 is still detectable. Scale bars are 10 μm. B) Scanning electron microscopy images of endothelial cell-denuded mesenteric resistance arteries show a comparable topography of the endothelial basement membrane in wild type, Tek-Cre::Lama5-/- and Lama4-/- arteries. The arrows indicate artificial ruptures due to preparation procedure. Scale bars are 100 nm. C) Intravital microscopic quantification of mesenteric arteries sizes in vivo show smaller diameters (-24.3%) in Tek-Cre::Lama5-/- arteries and larger diameters (+27.6%) in Lama4-/- arteries compared to wild type controls. Auto-fluorescence of the internal elastic lamina allowed a good approximation of mesenteric arteries lumen diameter. Scale bar is 100 µm. Data are means ± s.e.m from a minimum of 4 first order mesenteric arteries imaged at least in 4 mice per genotype. * P<0.05 unpaired t-test.
Figure 8Representative confocal micrographs show EphA2 (red), RSK (green: top) and GPRC5A (green: bottom) in frozen sections of HGSC patient tumors. S indicates the stroma. Scale bars: 50 μm. Indicated proteins and EphA2 phosphorylation were assessed by immunoblotting in patient-derived HGSC cells and CAFs. GPRC5A in earlier (top) and later passage (bottom) HGSC cells. See normalized GPRC5A relative to OCKI_p01 below the immunoblot images. Charts illustrate EphA2-pS897 inhibition (circles) and EphA2-pY588 increase (squares) by 10 µM BI-D1870 alone (unbroken line) or in combination with 0-10 µM cisplatin (dotted line) in the GPRC5Ahigh HGSC cells. See Appendix S7B for immunoblots. Cell viability of the GPRC5Ahigh OCKI_p02 and OCKI_p06 after treatment with a combination of 0-20 µM cisplatin and 10 µM BI-D1870 for 72 h. N = 3. Cell viability upon 72 h combinatorial treatment with 0-20 µM cisplatin and 25 µM LJH685 in patient-derived CAFs Cell viability upon 72 h combinatorial treatment with 0-20 µM cisplatin and 10 µM BI-D1870 in mesothelial cells (G; N = 3). Chart illustrates the changes of EphA2-pS897 and EphA2-pY588 by BI-D1870 alone and with cisplatin in mesothelial cells. Upon combination with 10 µM cisplatin for 72 h, 10 µM BI-D1870 decreased EphA2-pS897 and EphA2-pY588. See Appendix Fig S7J for immunoblot. Confocal micrographs of EphA2 (red) and GPRC5A (green) in primary OCKI_p06 patient cells cultured in 3D collagen for 4 d before 72 h treatment with or without 10 µM cisplatin. Scale bars: 20 μm.
Figure 5Growth curve of MRC-5 fibroblasts. Cells were grown until senescence and after three weeks of no growth detection, lentiviral infections containing either shp21CIP1 or shp21CIP1 + shRAP1 sequences were performed. Cells were harvested 15 days post infection (dpi). Data represent mean +/- SD of three biological replicates measured after 8 and 15 dpi. Expression of p21CIP1 and RAP1 after 15 dpi of cells transduced with shControl, shp21CIP1 or shp21CIP1 + shRAP1. Dividing cells (magenta) were identified by EdU staining (1 µM EdU for 24 h). The percentage of dividing cells after 15 dpi is shown (n = 3). Scale bar = 10 µm. Senescence-associated β-galactosidase (SA-β-gal) staining in senescent and post-senescent fibroblasts (1 and 15 dpi) is shown. Percentage of SA-β-gal positive cells is indicated for each condition. Three biological replicates were performed, with approximately 300 cells analyzed per condition. Scale bar = 50 µm. Senescent cells, transduced with shp21CIP1 or shp21CIP1+shRAP1 for 15 days, were stained with CytoCalcein violet (live cells), Apopxin Green (apoptotic cells) and 7-aminoactinomycin D (7-AAD) (late apoptostic/necrotic cells) and visualized by flow cytometry. Quantification of apoptotic cells from the conditions described in E. Data represent mean +/- SD of three biological replicates (**P < 0.001; two-tailed Student's t test).
Figure 6(A) The (A) NP proteins in the cell lysates were determined by Western blot analysis. (B, C) The virus RNA in the (B) culture medium and (C) cell lysates was quantified by real-time RT-PCR. The genetic information and sequence reference numbers for the 5 SARS-CoV-2 strains were summarized in Table EV1. Error bars represent the standard deviation (SD), n=3. Statistical analysis was performed by unpaired two tail t-test. ** P < 0.01, *** P < 0.001. Experiments were performed at least three times with similar results.
Figure 2(A) Average expression of Dppa2 and Dppa4 in a single-cell RNA-seq analysis of mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population. (B) Fraction of GFP+ cells in mESCs carrying a MERVL-GFP reporter and depleted of Dppa2 or Dppa4 using shRNAs specific to the transcripts or a control. Bars represent the average, error bars the SD; n = 3. ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test. (C) Dppa2, Dppa4, Dux and Zscan4 gene expression in mESCs carrying a MERVL-GFP reporter and depleted of Dppa2 or Dppa4 using shRNAs specific to the transcripts or a control. Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3. ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test. RNA-seq analysis of WT and Dppa2 (D) KO mESC clones. The dot plot displays the average gene expression of three independent clones from each cell type; n = 3. RNA-seq analysis of WT and Dppa4 (E) KO mESC clones. The dot plot displays the average gene expression of three independent clones from each cell type; n = 3. (F) Comparative expression by qPCR of Dux, two downstream targets of DUX (Zscan4 and Tdpoz4), and two DUX-independent targets of DPPA2 and DPPA4 (Mael and Tdrd1) in mESCs depleted of endogenous DPPA2 and DPPA4 and overexpressing ectopically DUX or GFP as a control. Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3 (G) Venn diagram representing the overlap of genes dowregulated in Dppa2 and Dppa4 KO compared to WT cells. (H) Comparative expression of DUX-dependent, DPPA2 and DPPA4-dependent, and the rest of the genes in mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population (unpaired t-test).
Figure 6A Flow cytometry analysis of cardiac troponinT (cTNT) expression was performed at day 60 post cardiac differentiation. 98% of cTNT+ cardiomyocytes (CM) were obtained after metabolic selection. B RT-PCR showing the expression of indicated genes in undifferentiated iPSCs and differentiated iPSC-CMs of BTHS patient TAZ10 and a healthy control. C Immunostaining of control (left) and BTHS iPSC-CMs at day 60 post differentiation for cardiac troponin T (cTNT) and Connexin 43 (Cx43). Scale bars: 50µm. D Quantitation of sarcomere organization. Left panel: Immunostaining of cTNT, MLC2a (myosin light chain) and a-actinin at day 60 post cardiac differentiation. Right panel: The bar graphs at the right panel represent the sarcomere regularity from 3 independent experiments. Error bars represent standard deviation for the comparison between control and BTHS patient TAZ10. Significance was analyzed using Student's T-TEST **P = 0.002 and ***P = 0.0006. Scale bars: 50 µm. E Measurement of cell surface areas. Cell surface area was measured in single cardiomyocytes (n = at least 30 measurements each group from N = 3 independent experiments each group) at day 60 post cardiac differentiation after immunostaining of sarcomeric a-actinin. Scale bars: 50 µm.
Figure 2Quantitative RT-PCR showing the mRNA expression levels of IL-1β, IL-18, ASC, CASP1 and NLRP3 in MDMs treated with P22077 (2.5 µM) and/or LPS (1 μg/mL; 4 hrs.), as indicated. Bars represent the mean fold increase from an untreated (UT) control Western blots showing the relative abundance of NLRP3, ASC, pro-caspase-1, pro- IL-1β, pro-IL-18, USP7 and USP47 in cell lysates of MDMs treated with P22077 (2.5 µM) and/or LPS (1 μg/mL) for 4 hrs., as indicated. β-actin is shown as a loading control. Blots are representative of at least 3 independent blood donors. IL-18 ELISA of supernatants from LPS-primed (1 μg/mL, 10 mins.) MDMs pre-incubated with either vehicle (0.1% DMSO) or P22077 (2.5 µM) for 15 mins. before treatment with nigericin (10 μM, 45 mins.). Bars represent the mean, ± S.D. n = 9 independent blood donors. Western blots of supernatants and cell lysates from MDMs as treated in C. Bands in the figure represent: Pro-IL-18 (24 kDa), mature IL-18 (mIL-18, 18 kDa) and β-actin as a loading control. Blots are representative of at least 3 independent blood donors. LDH-release from MDMs as treated in C. Bars represent the mean percentage of LDH release relative to the total cells lysed, ± S.D. n = 9 independent blood donors.
a, b) APG5-dependent punctate GFP–LC3 fluorescence in Bcl-2-transfected MEFs after etoposide treatment.figf4(a, b) APG5-dependent punctate GFP-LC3 fluorescence in Bcl-2-transfected MEFs after etoposide treatment. APG5+/+ and APG5−/− MEFs that were transfected with both GFP-LC3 and the indicated plasmids (Bcl-2 or vector) were incubated without or with 20 μM etoposide for 24 h, then examined by confocal fluorescent microscopy (a). The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells (b). (c) Accumulation of Beclin 1 and APG5-APG12 complex in Bcl-2-transfected MEFs after etoposide treatment. WT MEFs were transfected with the indicated plasmids, and were incubated without (NT) or with 20 μM etoposide for 24 h. (d, e) Occurrence of etoposide-induced 3-MA-inhibitable non-apoptotic death in Bcl-xL-overexpressing APG5+/+ MEFs, but not APG5−/− MEFs. APG5+/+ (open columns) and APG5−/− (closed columns) MEFs were transfected with the indicated plasmids (2 μg). After 24 h, transfected cells were incubated with 20 μM of etoposide in the presence or absence of 100 μM of zVAD or 10 mM of 3-MA for 24 h, and cell viability was measured by the CTB assay (d) and apoptotic nuclear morphology (e). Data are shown as mean ± s.d. (n = 4). (f) Overexpression of Bcl-xL does not sensitize Beclin 1-silenced MEFs to etoposide-induced 3-MA-inhibitable non-apoptotic death. WT MEFs with silencing of control GFP (open columns) or Beclin 1 (closed columns) were transfected with the indicated plasmids (1 μg). After 24 h, transfected cells were incubated with 20 μM of etoposide in the presence or absence of 10 mM of 3-MA for 24 h, and cell viability was measured by the CTB assay. n = 3.
Autophagy induction by overexpressed Atg14L.Figure 4.Autophagy induction by overexpressed Atg14L. A549 cells were transiently transfected with adenovirus harboring GFP-Atg14L or GFP-Atg14L4C4A. (a and b) The cells before starvation (a) or after starvation (b) were fixed and stained with anti-LC3 antibody. (c) The number of LC3-positive puncta per cell was counted, and mean ± SD values are presented. Bars, 10 µm.
Fig.Figure 6A-D) Wild-type (A, B) or nyv1∆ cells (C,D), which lack the vacuolar R-SNARE Nyv1, were labeled with FM4-64. Cells were immobilized on a chambered slide and subjected to FRAP experiments (C) Vacuole fission induced by hypertonic shift. FRAP experiments were performed before (0') or 15 min after addition of NaCl to 0.5 mol/l. The fluorescence images show the vacuoles inside a single cell. (B, D) Cells in S-phase were selected, which grow a daughter cell and therefore fission their vacuoles into a chain of tubulo-vesicular structures, part of which are transported into the daughter cell. FRAP experiment on vesicles in the inheritance structure were performed. Arrows indicate the bleaching area, dashed orange lines the cell cortex. Scale bar: 2 µm.
Localization and post-translational modifications of WASH1 during mitosis Phos-tag Western blot of MCF7-Her2-18 cells synchronised into distinct phases of the cell cycle showing the phospho-shift in WASH1 during mitosis.Figure 5Phos-tag Western blot of MCF7-Her2-18 cells synchronised into distinct phases of the cell cycle showing the phospho-shift in WASH1 during mitosis. Blue native PAGE Western blot showing shift in the size of WASH1 protein complex isolated from STLC treated prometaphase arrested cells when compared to cells forced to exit mitosis following Ro3306 treatment.
Figure 1Schematic of ssDNA curtain assay. Widefield images of RPA-mCherry-ssDNA molecules (magenta) in the presence of GFP-Rdh54 (green; 1, 3, 10 and 30 nM). Widefield images of Rad51-ssDNA molecules (unlabeled) in the presence of GFP-Rdh54 (green; 1, 3, 10 and 30 nM). Kymograph illustrating the dissociation of GFP-Rdh54 from Rad51-ssDNA; arrowhead highlights the time point at which free GFP-Rdh54 was flushed from the sample chamber.   Graph depicting the dissociation rate of GFP-Rdh54 from Rad51-ssDNA. Error bars represent s.d. of individual ssDNA molecules (N=25).   Kymograph showing the association of GFP-Rdh54 with a Rad51-ssDNA molecule; arrowhead highlights the injection time point for GFP-Rdh54.   Association rates of GFP-Rdh54 on individual Rad51 PSCs (N=44). Red lines represent the mean and s.d.
HR and FA pathway genes are expressed in the Wnt high compartment of the small intestine Immunohistochemical staining of sections of small intestine from C57BL/6J mice for DNA repair proteins shows that the expression of BRCA1, FANCD2 and RAD51 was high in the crypts (Wnt high compartment) whereas the villi did not show any staining.Figure 7Immunohistochemical staining of sections of small intestine from C57BL/6J mice for DNA repair proteins shows that the expression of BRCA1, FANCD2 and RAD51 was high in the crypts (Wnt high compartment) whereas the villi did not show any staining. Small intestinal adenomas from APCmin/+ mice displayed positive nuclear immunostaining for HR and FA pathway proteins BRCA1, FANCD2 and RAD51. The bottom panels are higher magnification images of the areas marked in the top panels. Expression of DNA repair genes correlates with the Lgr5 expression in mouse intestine: Gene expression analysis of sorted Lgr5 populations from the mouse intestine (dataset (GSE149311) shows that increased expression of known Wnt target genes Lgr5 and Axin2 correlates with Mybl2, as well as with Brca1/2, Rad51 and Fanc genes. The horizontal lines represent mean of replicates. PORCN inhibition reduces the expression of HR and FA pathway genes and Mybl2 in the normal mouse intestine. Total RNA was isolated from small intestines of C57BL/6J mice treated above in Figure 7C. Expression of DNA repair genes and Mybl2 was measured by qRT-PCR. Each data point represents an individual mouse. n = 3/group. The horizontal lines represent mean of replicates. PORCN inhibition reduces the expression of HR and FA pathway proteins in the normal mouse intestine. C57BL/6J mice were treated with vehicle or ETC-159 (60 mg/kg) for 3 days. Small intestines were sectioned and analyzed by immunostaining for BRCA1, FANCD2, RAD51 and phosphorylated-BRCA1.
Figure 2Zoom-ins into ABCE1-30S connections. Most interactions are salt-bridges or H‑bonds between ABCE1 residues and the rRNA phosphate backbone. The FeS cluster domain contacts rRNA h5 via R2 and K59, interacts with uS12 via S29 and R28, and contacts h44 by N17 and K15. The helix-loop-helix motif connects to rRNA h15 via R144 and E147. The positioning of the cantilever is stabilized by an interaction network of R2, I68 and N70 with N316 of hinge 1 and rRNA h5. Yeast survival of ABCE1 variants (S. solfataricus colored, S. cerevisiae in gray). Most residues connecting to 30S in the post-SC show a growth defect when exchanged for a small one (alanine) or a negative charge (glutamate). ++ no effect, + growth defect, - lethal. The cantilever link forms salt-bridges of E74 and E76 with NBD1 residues K89 and R293, respectively.
Figure 1Cartoon depicting the mutagenesis strategy, including the genomic target locus, as well as the structure of targeting vector. Arrows indicate location of primers used for genotyping. Restriction enzyme diagnostic test for the presence of the R749H mutation. Sequence trace of cDNA showing the presence of the heterozygous and homozygous R749H mutation. qRT-PCR with primers specific to both wild-type and mutant RNAPII (left panel) or to the mutant form of RNAPII (right panel), confirming that only the slow version of RNAPII is expressed in homozygous slow/slow ESCs. The sequences of the respective forward primers are shown. The "WT/slow allele" primer is complementary to the sequence in exon 14 upstream of the mutation. The "slow allele only" primer has its 3' end matching the mutated codon 749 and does not anneal to the WT DNA sequence. The mean ± SEM is plotted, n=2.