EMBO/sd-character-level-ner huggingface dataset:
a, Analysis of BCL2 phosphorylation (detected by anti-BCL2 immunoprecipitation and autoradiography of 32P-labelled cells) and beclin 1 co-immunoprecipitation with BCL2 in wild-type (WT) or BCL2 AAA MEFs grown in normal media or subjected to 4 h Earle’s balanced salt solution (EBSS) starvation.f2a, Analysis of BCL2 phosphorylation (detected by anti-BCL2 immunoprecipitation and autoradiography of 32P-labelled cells) and beclin 1 co-immunoprecipitation with BCL2 in wild-type (WT) or BCL2 AAA MEFs grown in normal media or subjected to 4 h Earle's balanced salt solution (EBSS) starvation. p-BCL2, phospho-BCL2. b, Quantification of GFP-LC3 puncta (autophagosomes) in MEFs of indicated genotype in normal growth conditions or starvation conditions. Data represent mean ± s.e.m. for 100 cells per well of triplicate samples per condition. Similar results were observed in three independent experiments. c, Representative images of GFP-LC3 puncta (autophagosomes) in skeletal and cardiac muscle of GFP-LC3 wild-type and GFP-LC3BCL2 AAA mice before exercise, after 80 min exercise, or after 75% of maximal exercise capacity. Scale bar, 20 µm. d, Quantification of data (mean ± s.d. of 4 mice per experimental group) in c. e, Western blot analysis of LC3-I/II and p62 levels in indicated tissue from mice of indicated genotype at rest (−) or after maximal exercise (+). Skeletal and cardiac indicate skeletal and cardiac muscle, respectively. f, Co-immunoprecipitation of beclin 1 with BCL2 in muscle tissue from mice of indicated genotype at rest (−) or after 30 min of exercise. WCL, whole cell lysates. NS, not significant. *P  0.05, **P  0.01, ***P  0.001, one-way ANOVA for comparison between groups; †P  0.001, two-way ANOVA for comparison of magnitude of changes between different groups in mice of different genotypes.
Loss of p53 hastens EMT in NFATc1‐driven pancreatic cancer modelsGenome‐wide expression and GSEA analysis show p53‐enriched signatures with “EMT” and “stem cell transcript” in KNC tumor cells.Figure 2A Genome‐wide expression and GSEA analysis show p53‐enriched signatures with "EMT" and "stem cell transcript" in KNCtumor cells. B Heat map showing p53‐dependent regulation of "EMT"‐ and "stemness"‐related genes in KNCtumor cells. Fold change relative to control cells is displayed in a green red color scheme for selected genes with FClog2 1.0 or FClog2 > −1.0. C Statistical analysis of wound healing assay performed in primary KNCtumor cells following p53 depletion. Time‐lapse analyzer software was used to quantify the wound closure rate. Means ± SD are shown from one out of three independent experiments. D Western blot analysis revealing protein expression levels upon p53 depletion in KNCtumor cells. E X‐fold mRNA expression of EMT‐related marker genes after p53 depletion in KNCtumor cells. Means ± SD from at least three independent experiments are shown. Asterisks display significance (*P 0.05). F Representative protein expression of EMT inducers in pancreatic lysates prepared from KNCmice with different tumor gradings.
Figure 5Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (A) Body weight. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance. Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (B) Body composition. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance. Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (C) Sirius Red staining of representative embedded BAT, sWAT and vWAT from mice on HFD and quantification of adipocyte areas (~1000 adipocytes/genotype). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance. Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (D) IPGTT after 12 weeks of diet and AUC. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance. Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (E) ITT after 13 weeks of diet and AUC Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance. Seven-week old CTRL and FAKO (Mllt4-/-) mice were given high-fat diet (HFD) for 16 weeks (n=5-6). (F) normalized to basal insulin. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance. Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (G) Plasma insulin levels 5 min after an IPGTT performed after 18 weeks of diet. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.
Figure 3A Measurement of free ABA levels in the third and fourth rosette leaves from 21-day-old transgenic lines with altered CDF4 expression after estradiol induction for three days (once every day). Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test. B, C qPCR analysis of ABA synthesis and signaling-related genes in Col-0 and 35S::CDF4 plants. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t-test. D, E Relative expression of NCED2 and NCED3 in 14-day-old CDF4GR transgenic plants treated with 20 μM β-estradiol or mock treatment for 0, 2, or 4 h. The expression of the corresponding genes in mock-treated plants was set to 1.0. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test. F, G Relative expression level of NCED2 and NCED3 in 14-day-old CDF4GR transgenic plants treated with 20 μM DEX, 100μM CHX, DEX plus 100μM CHX, or mock. The gene expression in mock-treated plants was set to 1.0. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test. H Schematic diagram indicating the locations of six DOF motif clusters (D1 to D6) in the NCED2 and NCED3 gene promoters. I ChIP-qPCR analysis of the ability of CDF4 to bind to the promoters of NCED2 and NCED3. An anti-HA monoclonal antibody was used for DNA immunoprecipitation from three-week-old pER8::CDF4-HA transgenic plants. Black bars indicate the enrichment fold-changes normalized to that of ACT2. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t-test. Transient dual-luciferase reporter assay. The pGreenII-0800 LUC construct containing the CDF4 promoter and the p62-SK construct with or without the CDF4 coding region and CDF4-VP16 were transiently co-transformed into Col-0 protoplasts. Firefly luciferase (LUC) and Renilla luciferase (REN) activities were measured after culturing the protoplasts under low light conditions for 16 h. The ProNCED2/3:LUC /Pro35S:REN ratio represents the relative activities of NCED2 and NCED3 transcription. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t-test. K Transient dual-luciferase reporter assay. The pGreenII-0800 LUC construct containing the CDF4 promoter and the p62-SK construct with or without the CDF4 coding region and CDF4-VP16 were transiently co-transformed into Col-0 protoplasts. Firefly luciferase (LUC) and Renilla luciferase (REN) activities were measured after culturing the protoplasts under low light conditions for 16 h. The ProNCED2/3:LUC /Pro35S:REN ratio represents the relative activities of NCED2 and NCED3 transcription. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t-test.
Arrangement of the eIF3c-NTD in human and yeast 43S PICs.Figure 6A Cryo-EM map obtained after focused sorting of the human 43S PIC on the TC: when low-pass filtered at 6 Å, it shows the density of almost-complete eIF3c-NTD in the ISS. B Cryo-EM map of the yeast 43S PIC low-pass filtered at 6 Å. C, D Model for human eIF3c in the TC-containing 43S colored in rainbow (C) and scheme of the alignment between human and yeast eIF3c sequences, colored accordingly (D). The eIF1-interacting stretch present in the N-terminus of S.c. eIF3c shows 32.0/56.0% sequence identity/similarity with an insert C-terminal of the conserved 4-helix bundle conserved in mammals. E Zoomed view highlighting the position of the eIF3c NTD and eIF1 in the 40S ISS. F Molecular model for the 4-helix bundle interacting with 40S rRNA and r-proteins.
Figure 8In wild-type pro-B cells, IL-7-mediated activation of the IL-7 receptor, consisting of the IL-7Rα and Cγ chains, leads to phosphorylation and activation of JAK1 and JAK3. These active kinases phosphorylate STAT5, which results in the formation of parallel STAT5 dimers that are transported across the nuclear membrane by importins. In the nucleus, STAT5 binds to DNA and regulates genes involved in cell survival, proliferation and metabolism (Villarino et al., 2017). Phosphatase-mediated inactivation of the DNA-bound STAT5 complex disengages the dimer from the DNA, followed by formation of the anti-parallel STAT5 dimer and its export via nuclear exportins to the cytoplasm. In Pax5Jak2/+ B-ALL cells, cytoplasmic STAT5 is similarly activated by IL-7R signaling and transported across the nuclear membrane. In the nucleus however, the constitutively active (ca) PAX5-JAK2 protein antagonizes the action of phosphatases by re-phosphorylating STAT5 and thus maintaining active STAT5 levels, leading to sustained expression of STAT5 target genes, which promotes leukemogenesis.
Figure 2(A) Selected frames from time-lapse imaging experiments (see Movies EV1 and EV2) showing two dividing cells transfected with either control (CTRL) or CITK-specific siRNAs, respectively. Note one of the two daughter cells in the lower panel (red arrow) going out of focus as a consequence of oblique division.(B) Graphical representation of vertical and oblique divisions. The cleavage plane is indicated by a black dashed line.(C) Quantification of divisions showing uneven timing of daughter cell flattening onto the substrate after mitosis (oblique division) in CITK siRNA treated HeLa cells compared to control (n > 50 cells, 3 independent experiments). (D) Control or CITK-depleted cells were immunostained for γ-tubulin (red) and DNA (blue) and imaged in z (0.3 µm-thick sections). Upper panel: maximum intensity projections of confocal z-stacks are shown. Lower panel: cross-section (XZ) through the two poles of the same cell.(E) Distribution of spindle angles (°) in control and in CITK-depleted cells. The values represent the angles between the axis crossing the two poles of metaphase spindles and the coverslip. (n ≥ 150 cells, 6 independent experiments). (F) Quantification of spindle angles average in control cells and in cells depleted of CITK, ASPM or co-depleted of the two proteins (n ≥ 150 cells, in at least 3 independent experiments).
PI3KC2β inactivation in the non-hematopoietic compartment reduces cerebral infarction and edema in vivo and preserves endothelial barrier integrity in vitro.Figure 4Representative photographs of coronal brain sections and graph quantification of infarct volume (dashed white line) measurements in bone marrow (BM) chimeric mice 24 hours after tMCAO. WT>WT: transplantation of WT BM into WT hosts; C2βD1212A>WT: transplantation of C2βD1212A/D1212A BM into WT hosts; C2βD1212A>C2βD1212A: transplantation of C2βD1212A/D1212A BM into C2βD1212A/D1212A hosts; WT> C2βD1212A transplantation of WT BM into kinase-dead hosts. Data represent mean ± SEM; n=13-16 mice per group; *p<0.05 **p<0.01; ***p<0.001 vs WT>WT, Mann-Whitney test or unpaired t-test, as accordance. edema volume in bone marrow (BM) chimeric mice 24 hours after tMCAO. WT>WT: transplantation of WT BM into WT hosts; C2βD1212A>WT: transplantation of C2βD1212A/D1212A BM into WT hosts; C2βD1212A>C2βD1212A: transplantation of C2βD1212A/D1212A BM into C2βD1212A/D1212A hosts; WT> C2βD1212A transplantation of WT BM into kinase-dead hosts. Data represent mean ± SEM; n=13-16 mice per group; *p<0.05 **p<0.01; ***p<0.001 vs WT>WT, Mann-Whitney test or unpaired t-test, as accordance. C Sh-Control or sh-PI3KC2β hCMEC/D3 cells were cultured on Transwell and stimulated with TNFα over the time. Trans-endothelial electrical resistance (TEER) was measured with a voltohmeter Millicell ERS-2 at the indicated time. Data are shown in percentage of control at t0 ± SEM (n=4 independent experiments). ***p<0.001; **p<0.01; *p<0.05, significance differences from control, 2-way ANOVA with Bonferroni post-test.
Figure 2(A) Phylogenetic analysis between mouse Tnfaip2 and planarian Smed-exoc3. Note that Tnfaip2 emerged in vertebrates before the split of cartilaginous and bone fish since both lineages carry \ the Tnfaip2 gene. Callorhinchus is a representative of cartilaginous fishes, while Danio and Nothobranchius are representatives of bony fishes. Note, that branch lengths proportionally reflect the minimal number of substitutions along the branch. The longer branch length of Tnfaip2 vs. Exoc3 in vertebrate species suggests that Tnfaip2 has undergone sub-functionalization. The species with both Tnfaip2 and Exoc3 are indicated, other species in the lower part only have Exoc3, which is not indicated. (B) Experimental scheme for injections of planarians (S. mediterranea) with dsRNA against Smed-exoc3 or control injections (gfp(RNAi)). (C) RT-qPCR analysis of Smed-exoc3 mRNA expression of total RNA (whole body-derived) from exoc3(RNAi)-treated planarians compared to control-injected planarians on day-38 of the injection scheme [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test); data were analyzed by one-sided t-test]. (D) Representative photographs of exoc3(RNAi)-treated planarians vs. control planarians at day-38 of the injection protocol as shown in panel B (n=4 experimental replicates with 6-10 biological replicates per experiment, scale bar: 0.5 mm). Number ratios in the photographs depict the total number of animals showing the indicated phenotype.
Figure 5A) Partial amino acid sequences of the hRSV F (red) and hMPV F (blue) antigenic site IV (Lopez et al., 1998;McLellan et al., 2010;Wu et al., 2007). Partial sequences of the F-410 and F-411 chimeras, color coded to indicate the origin of their antigenic site IV residues. Amino acids shared by hMPV F and hRSV F are shown in purple. The remaining sequences of F-410 and F-411 are derived from prefusion hRSV F. B) Surface representation of the hRSV F structure stabilized in the prefusion conformation, as reported (McLellan et al., 2013a). One of the protomers is shown as a red ribbon. Antigenic site IV is magnified and colored as in (A) for the F-410 and F-411 chimeras. C) ELISA binding results of mAbs specific for hRSV F (left panels) or hMPV F (right panels) with the proteins indicated at left. mAbs specific of hRSV F bind to epitopes of antigenic site Ø (D25), antigenic site II (Mz and 47F) or antigenic site IV (101F and 107F). mAs specific of hMPV F bind to epitopes of antigenic site II (MF14), antigenic site IV (MF11, MF16 and MF29) or an epitope shared by hRSV F and hMPV F (MPE8).
PBX1 is present in mDAn.Figure 1(A) Tru-Seq RNA sequence analysis of E12.5 midbrain floor-plate (mFP), midbrain roof-plate (mRP), anterior (A, adjacent anterior FP) and posterior (P, adjacent posterior FP). Pbx1 is enriched in the midbrain FP, together with Th and Pitx3. Lower levels of Pbx3 are also expressed in the mFP. Gdf7 and Wnt1 are restricted to the mRP at E12.5. (B) Pbx1 is expressed in the intermediate zone (IZ) and marginal zones (MZ), but not the ventricular zone (VZ), of the mFP at E12.5, as detected by in situ hybridization. (C) PBX1 is first detected in the ventro-lateral part of the LMX1a+ domain at E10, preceding the birth of the first (TH+) mDA neurons at E10.5. At E12.5, PBX1 is present in all mDA neurons, but not all PBX1+ cells are TH+. White boxes indicate the area showed in higher magnification (right). (D) At E11.5, PBX1 protein defines a subpopulation of NURR1+ neuroblasts and labels all NURR1+TH+ mDA neurons. (E) PBX1 co-localizes with PITX3 and is also detected in a subpopulation of NURR1+PITX3- postmitotic neuroblasts at E12.5. Higher magnification revealed three different populations of postmitotic cells: primary neuroblasts (NURR1+PBX1A-PITX3-cells, green), secondary neuroblasts (NURR1+PBX1A+PITX3-cells, yellow/orange) and tertiary neuroblasts/mDA neurons (NURR1+PBX1A+PITX3+cells, white). Nuclear staining, Dapi (4′,6-diamidino-2-phenylindole, blue). All scale bars, 20µm.
Figure 5(A) A549 cells of the indicated genotypes were either left untransduced or transduced with a lentiviral vector expressing PIDD1-V5 in its wild type form. Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting. N = 2 independent experiments. (B,C) A549 cells of the indicated genotypes were either left untransduced (mock), or transduced with lentiviral vectors expressing PIDD1-V5 in its wild type form or carrying the indicated point mutations. Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting. N = 2 independent experiments. (D) Fluorescence micrographs of A549 cells of the indicated genotypes. Cells were either left untransduced (mock) or transduced with PIDD1-V5 lentiviral vectors carrying the indicated point mutations, and stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.
Figure 6(A) Schematic of the results from the drug screening with compounds targeting GPCR signaling. MDA-MB-468 cells were treated with the compounds of the GPCR-targeted library at three different concentrations (0.1, 1 or 10 μM) in combination with vehicle, GDC0941 450 nM or lapatinib 1 μM. The pS6 signal was measured by IF after 24 hours of treatment and normalized to DAPI. Z scores of the normalized fluorescence values for each drug measured in the presence of vehicle, GDC0941 or lapatinib are reported on the y axis or on the left or right x axis of the dot plot, respectively. Readings acquired following treatment with the three different concentrations of the GPCR-targeted drugs are reported in 3 different colours. Dots corresponding to vorapaxar treatments at the three different concentrations are highlighted in the plot. The values reported are mean of a biological triplicate of the experiment. (B) MDA-MB-468 were treated for 24 h with vehicle, vorapaxar (10 μM), GDC0941 (1 μM) or lapatinib (1 μM) alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies. (C) MDA-MB-468 parental cells or GNB2 KO clones were starved and treated with scramble or PAR1 activating peptide for 5 minutes, alone or in combination with vorapaxar. The cell lysates were probed with the indicated antibodies. Phospho-AKT, pan-AKT, phospho-ERK1/2 and pan-ERK1/2 bands were quantified by the use of ImageLite software: the ratio of phospho-AKT to pan-AKT and phospho-ERK1/2 to pan-ERK1/2 normalised to the control peptide-treated conditions for WT and GNB2 KO cells (first and forth lanes, respectively) is shown. (D) p-S6/loading control signal from western blots experiments in which a panel of six TNBC PTEN-null cell lines and HCC70 cells that acquired resistance to AZD8186 or MK2206 were treated with vehicle, vorapaxar (10 μM), GDC0941 (1 μM) or lapatinib (1 μM) alone or in the indicated combinations. The values were normalised to vehicle treatment for all cell lines. Mean of 2 independent experiments ± SD. P values calculated by two-tailed paired student t-test *P=0.0235 and **P=0.002. Cell lines used in the experiments were: MDA-MB-468, HCC70, HCC1937, HCC38, HCC1395, BT-549, HCC70 AZD8186-resistant and HCC70 MK2206-resistant. (E) Long-term proliferation assay of MDA-MB-468 cells treated with vorapaxar (5 or 2.5 μM), GDC0941 (1 μM) or lapatinib (1 μM), alone or in the indicated combinations. Cells were treated for two weeks and stained by crystal violet. One representative experiment of three is shown. (F) Schematic of the network controlling the phosphorylation of AKT and S6 in PTEN-null TNBC cells. EGFR and PAR1-βγ subunits of G protein signal in parallel to p110β and AKT. The two arms of the pathways are inter-connected by feedback compensation mechanisms that act when any of the two signals is perturbed. Combined targeting of both the signaling arms results in a sustained inhibition of the downstream pathway. Erlotinib (EGFR inhibitor) and AZD8186 (p110β inhibitor) are highlighted in the figure as these drugs proved effective in in vivo experiments.
A minority of CTL-tumor cell interactions results in target cell apoptosis in vitroOVA+Eµ-myc B cells expressing a FRET-based reporter for caspase 3 activity were labeled with the calcium-sensitive dye Indo-1, incubated with OT-I CTLs and subjected to live imaging.Figure 2(A-B) Calcium responses were recorded over time in (A) unconjugated tumor cells or in (B) tumor cells forming a conjugate with a CTL (during the duration of the contacts). Each line corresponds to an individual tumor cell and each square to a time point. Squares are colored in magenta upon calcium elevation or in blue otherwise. Apoptotic events (detected by caspase 3 activity) are indicated by an overlaid green dot. Data is representative of three independent experiment. (C) The number of calcium fluxes in individual tumor cells is plotted for unconjugated tumors or conjugated tumors. Values were normalized to 100 min. Each dot represents one tumor cell. Bars represent mean values. (D) The cumulated period of calcium signals was recorded for individual unconjugated or conjugated target cells and expressed as percentage of total time. Each dot represents one tumor cell. Bars represent mean values. Distinct tumor cell outcomes in response to CTL attack. Three categories were defined: no hit (no calcium response in the target cell), sublethal hit (transient calcium response and no caspase 3 activity), lethal hit (prolonged calcium response and induction of caspase 3 activity). (E) Pie chart representing the percentage of each category. Distinct tumor cell outcomes in response to CTL attack. Three categories were defined: no hit (no calcium response in the target cell), sublethal hit (transient calcium response and no caspase 3 activity), lethal hit (prolonged calcium response and induction of caspase 3 activity). (F) Time-lapse images (left) of a representative CTL-tumor cell conjugate in each of the three categories are shown together with the corresponding quantification (right) of calcium response and caspase 3 activity over time. T cells are delineated by white dashed lines. Scale bar, 10 µm. Data were pooled from three independent experiments. (**p<0.01, unpaired t‑test).
Figure 2Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing the ubiquitination pattern of RNF8-WT and RNF8-RING* variant, under siRNA-mediated luciferase (siLuc) or p97-depleted conditions (sip97). Western blot analysis of CHX chase kinetics in HeLa cells showing the degradation kinetics of RNF8 and inhibition of RNF8 degradation by simultaneous proteasome inhibition (MG132, 10µM).   Graph represents the quantifications of (B) (ns; not significant, P>0.05, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).   Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing hyper-ubiquitination of RNF8 after proteasome inhibition (MG132, 10µM for 6h). Western blot analysis of CHX chase kinetics in U2OS cells, comparing the degradation rate of Flag-RNF8-WT and Flag-RNF8-RING*. Endogenous RNF8 was depleted by shRNF8 targeting only endogenous RNF8.   Graph represents the quantifications of (E) (ns P>0.05, ****P<0.0001; two-way ANOVA, n=3, mean +SEM).