EMBO/sd-character-level-ner huggingface dataset:
Figure 4All diagrams are aligned at the left and at the same scale, using the full-length E protein (Ev, top line) and sE401v (second line), derived from virions, as reference. Recombinant constructs are shown below the dashed line: sE400r, sE401r, sE404r (containing 6 N-terminal amino acids of H1), sE412r (containing H1), sE419r (containing H1 and H2), sE428r (containing H1, H2 and CS) and sE448r (containing the whole stem). Abbreviations EK and StT indicate heterologous sequences corresponding to an enterokinase-specific cleavage site and a strep-tag used for affinity purification, respectively, added at the C-terminal end of each construct. Trimer stability expressed as percentage of the fraction detected in the trimer peak and normalized with respect to the corresponding Ev fraction in sedimentation analyses after incubation at 70°C relative to 37°C (100%). Data are from at least three independent experiments.
Figure 1(A) Colony morphology and alkaline phosphatase (AP) activity of ES cells upon KD of Yap1 and Pou5f1. KD1 and KD2 indicate two different shRNA sequences tested. All the following cell morphology and AP staining pictures were taken two passages (4 days) after lentivirus infection unless otherwise stated. (B-C) mRNA expression levels of Pou5f1, Nanog, Sox2, Esrrb (B), and Yap1 (C) upon KD of Yap1. All the following mRNA samples were harvested 4 days after lentivirus infection while passaged every 2 days unless otherwise stated. Data are represented as mean SD. (D) Protein levels of Yap1, Pou5f1, and Nanog upon KD of Yap1. All the following protein samples were harvested 4 days after lentivirus infection while passaged every 2 days unless otherwise stated. (E) Colony morphology and AP activity of mouse embryonic stem cells (ESC) and three Yap1 KO clones (KO1-KO3). (F) mRNA levels of Pou5f1, Nanog, Sox2, and Esrrb upon KO of Yap1. Data are represented as mean SD. (G) Protein levels of Yap1, Pou5f1, and Nanog in Yap1 KO clones. (H) A heatmap showing relative mRNA expression levels of 3,605 genes differentially expressed (> 2-fold) between ES cells and differentiating ES cells (dESC). Genes were sorted by the fold changes of gene expression between dESC and ES cells. Corresponding gene expression profiles obtained from Yap1 KO1, Yap1 KO2, and Yap1 KD cells are also shown. (I) mRNA expression levels of lineage-specific marker genes upon KD of Yap1. dESC were used as control cells. (J) A heatmap showing Pearson correlation coefficients of gene expression profiles obtained from ESC, control virus infected ES cells (Control), dESC, Yap1 KD cells, and Yap1 KO cells. (K) Relative average module activities (Core and PRC) in Yap1 KD1 cells, KO cells and dESC. Module activities were normalized by the data obtained in ES cells. Data are represented as mean SEM. (L) Scatter plots showing log10 (FPKM) values of genes in Yap1 KD1 cells and Control (upper left panel), dESC and ESC (bottom left panel), and Yap1 KO cells and ES cells (right two panels). Pearson's correlation coefficients (R2) are indicated. 'FPKM' indicates Fragments Per Kilobase of transcript per Million fragments mapped.
a) Immunoblot detection of the phosphorylation status of AMPKα, ACCα, TSC2 and p70S6K in HCT116 cells.figf3(a) Immunoblot detection of the phosphorylation status of AMPKα, ACCα, TSC2 and p70S6K in HCT116 cells. Hypophosphorylation of p70S6K and hyperphosphorylation of AMPKα, ACCα and TSC2 was detected in p53−/− HCT116, compared with WT HCT116 cells after culture in complete medium (n = 5). (b) Quantification of GFP-LC3vac cells in WT and p53−/− HCT116 cells transfected with siRNAs specific for the catalytic α1 and α2 subunits of AMPK (mean ± s.d. of 3 independent experiments; *P 0.05). The inset in b demonstrates the efficiency of siRNA-mediated downregulation of AMPKα1 and AMPKα2 , as assessed by immunoblot analysis (n = 3). Note that the antibody recognizes both AMPKα1 and AMPKα2. (c) Immunoblot detection of ACCα phosphorylation in WT and p53−/− HCT116 cells subjected to knockdown of AMPKα1 and AMPKα2 (n = 5); Co, control. (d) Quantification of GFP-LC3vac cells (means ± s.d., n = 3 separate experiments) in WT HCT116 and p53−/− HCT116 treated with PFT-α and/or rapamycin.
Inhibition of Zic protein activity differentially affects spinal cord and hindbrain cell fates.Figure 2One-cell stage embryos were injected animally with the Zic1-MO (25/40 ng). Total RNA was isolated from ten control embryos at neurula stage 18 (lane 2) and ten embryos from each Zic1-MO injected group (lanes 3-4). Neural AP markers were examined by sqRT-PCR: forebrain (xanf1, otx2), hindbrain (krox20, hoxb1, hoxb3), spinal cord (hoxb9, cdx1-4) and panneural (soxd). Ef1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1). One-cell stage embryos were injected animally with mRNA encoding Zic5 dominant-negative (0.5 ng) protein. Total RNA was isolated from five control embryos at neurula stage 18 (lane 1) and five embryos from the injected group (lanes 2). Neural AP markers were examined by sqRT-PCR: hindbrain (krox20) and spinal cord (cdx1-4). Ef1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 3). One-cell stage embryos were injected animally with the Zic1-MO (25 ng). Embryos underwent in situ hybridization. Expression of the panneural sox2 marker was normal, the forebrain marker xanf1 was expanded in 80% of the embryos, the hindbrain representative marker krox20 was reduced in 80% of the embryos and the representative spinal cord marker cdx4 was expanded in 90% of the embryos. N = 23 embryos were per marker.
Figure 1WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Lethality was followed-up. LPS-treated mice (n=5) are depicted as black circles and DEX/LPS-treated mice (n=5) are depicted as white circles. P-values for survival curves were analyzed with a Fisher's exact test. WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Blood samples were taken 2 hours after LPS and TNF levels were measured in serum (n=4-5). WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Three hours after LPS challenge, dextran labeled with FITC was administered by oral gavage. Five hours after gavaging, blood plasma was collected and fluorescence was measured as a degree of intestinal permeability (λexc/λem = 488/520 nm) (PBS: n=3, LPS and DEX/LPS: n=5). RNA sequencing (RNAseq) results of IECs of WT mice (n=3) treated with PBS or 12.5 mg/kg LPS for 6 hours. Heatmap with selection of LPS-induced genes. Color key shows the log2 of normalized counts. RNA sequencing (RNAseq) results of IECs of WT mice (n=3) treated with PBS or 12.5 mg/kg LPS for 6 hours. HOMER motif analysis of LPS-induced genes. Motifs with the highest rank and their p-value and q-value are displayed. WT mice (n=5) were treated with PBS or 12.5 mg/kg LPS with or without 10 mg/kg DEX pre-treatment. Ileum was isolated and gene expression was measured via qPCR.
with an increased nuclear translocation in vitro.Figure 1(A, B) Overexpression of wildtype full-length human tau (hTau, also termed tau441 or tau40 or tau2N4R) induced significant alterations of 9 transcription factors screened by using Transcription Factors Activation Profiling Plate Array II, in which 96 transcription factors (Appendix Table S1 and S2) were monitored. The empty vector was transfected as a control (Ctrl). (C-F) Expression of hTau (probed by HT7) increased total and the phosphorylated STAT1 at Tyr701 (pY-STAT1) in cell whole extracts (C, D) and the nuclear fraction (E, F) measured by Western blotting (n=4). Data information: Data were presented as mean ± SD (Mann-Whitney test). *, p<0.05 vs Ctrl. (G) The representative immunofluorescent images and quantitative analysis show significantly increased STAT1 signal in the nuclear fraction of HEK293 cells with overexpression of hTau compared with the empty vector control (eGFP) (n=5). Scale bar, 10 μm. Data information: Data were presented as mean ± SD (Mann-Whitney test). , **, p<0.01 vs Ctrl. (H) Overexpression of hTau most significantly increased STAT1 monomer and dimer formation in nuclear fraction (Nu) measured by Western blotting. (I) Overexpression of hTau increased STAT1 activity in HEK293 cells detected by luciferase assay (n=4). Data information: Data were presented as mean ± SD (Mann-Whitney test). *, p<0.05 , ***, p<0.001 vs Ctrl. (J) Overexpression of hTau increased STAT1-DNA binding activity in HEK293 cells measured by electrophoretic mobility shift assay (EMSA). *, indicates STAT1/DNA complex.
Aged lungs exhibit persistent inflammatory responses following influenza infection.Figure S1.
Figure 3A. LNCaP cells silenced for SDHA or SDHB showed approximately 20-30% longer AR half life compared to ENZA alone in the presence of 50 µg/ml cycloheximide(left panel). Right panel shows quantification of AR protein normalized by loading control. B. Co-silencing of SDHB and Hsp27 reversed siSDHB-mediated AR protein upregulation in LNCaP cells. C. SDH-repressed LNCaP cells were able to maintain higher proliferation rate particularly during ENZA stress with or without Hsp27 silencing in real time cell proliferation assay. Data information: ENZA: enzalutamide. Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA following Tukey's test for panels C (end points) . *, p<0.05, **, p<0.01 and ***, p<0.001 compared between groups D. Hsp27 silencing in LNCaP cells showed dramatically high levels of cleaved-PARP compared to ENZA, which were rescued to some extent by SDH-repression. E. Treatment with p-p38 inhibitors: SB203580 (10 µM) or Genistein (20 µM) for 24 h reduced AR upregulation induced by SDH repression in LNCaP cells (left panel). Bar graph in the right panel shows band intensity normalized to loading control. Data information: ENZA: enzalutamide. Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA following Tukey's test for panels E . *, p<0.05, **, p<0.01 and ***, p<0.001 compared between groups
Figure 1A. Binding of ubiquitin chains to immobilized GST-fusion proteins. Indicated proteins were purified from E. coli and incubated with equal amounts of preformed fluorescently labeled ubiquitin chains, which were either K48- or K63-linked. The fraction of ubiquitin chains interacting with the bait protein was analyzed by SDS‑PAGE and fluorescence scan. Total fluorescence was quantified per lane and normalized to the first lane (rel. fluo.). Asterisk (*) indicates lane from the same gel that was cropped and moved. B. A structural model of Ube2K in complex with two ubiquitin molecules The UBC domain associates via its active site cysteine with the side chain of K48 in the immediate acceptor ubiquitin molecule (UbA‑prox). A second ubiquitin molecule (UbA‑dist) binds the UBA domain through the conserved hydrophobic patch. Selected atoms were highlighted as spheres: C-atom in the carboxy group (CC) of UbA‑prox-G76, S‑atom in E2 active site Cys, CC in UbA‑dist-L71, N-atoms (blue) in UbA‑prox of ε‑amino groups of lysine residues (Nε) and in the backbone of M1 (Nα). A structure of Ubc1 was superimposed onto the model Key residues were highlighted as sticks for the binding interfaces (C) between the UBC domain and UbA‑prox, Additionally, amino acids whose side chains contribute to the UBC/UBA binding interface were determined and classified into "hot spot" or "contributing" residues using the algorithm SpotOn -E. A structure of Ubc1 was superimposed onto the model Key residues were highlighted as sticks for the binding interfaces (D) between the UBA domain and UbA‑dist, Additionally, amino acids whose side chains contribute to the UBC/UBA binding interface were determined and classified into "hot spot" or "contributing" residues using the algorithm SpotOn A structure of Ubc1 was superimposed onto the model Key residues were highlighted as sticks for the binding interfaces (E) between the UBC domain and UBA domain. Additionally, amino acids whose side chains contribute to the UBC/UBA binding interface were determined and classified into "hot spot" or "contributing" residues using the algorithm SpotOn F. Interatomic distances from L71‑CC in UbA‑dist to G76-CC in UbA-dist or to putative attachment sites in UbA-prox were measured for various known ubiquitin conformations. The six closest attachment sites are listed in ascending order.
Biochemical analysis of TFEB-mediated reduction of pTau A Western blot analysis of detergent-free (Soluble) and sarkosyl-insoluble (Insoluble) preparations of total (total Tau), unphosphorylated (Tau1) or CP13- (S202/T205) and PHF1-positive pTau species in 4-month-old Tau or Tau + TFEB mice with P0 injection.Figure 2AWestern blot analysis of detergent−free (Soluble) and sarkosyl−insoluble (Insoluble) preparations of total (total ), unphosphorylated (Tau1) or CP13− (S202/T205) and PHF1−positive pTau species in 4−month−old Tau or Tau + TFEBmice with P0 injection. γ−tubulin was used as a loading control. B, C Quantification of relative band intensities in soluble (B) and insoluble (C) fractions. n = 3 mice/genotype/treatment group. CP13 and PHF1 are significantly reduced in both soluble and insoluble fractions (Student's t−test, ***P = 0.00072 and **P = 0.004, for B; and **P = 0.007 and **P = 0.009 for C). D Western blot analysis of total Tau, unphosphorylated Tau (Tau1) and CP13‐ or PHF1‐positive pTau levels in response to doxycycline induction (Tau) and/or TFEB‐FLAG transfection (TFEB) in T40PL cell line. Ctrl: untreated; Tau: 0.5 μg/ml doxycycline treated for 48 h; TFEB: TFEB transfected for 48 h; Tau + TFEB: combined TFEB transfection and DOX treatment. FLAG: anti‐FLAG antibody blotting for TFEB expression. γ‐tubulin was used as a loading control. E Quantification of relative band intensities. CP13, PHF1 and total Tau are significantly reduced by TFEB transfection (Student's t−test, n = 3, **P = 0.0012, 0.0040, and 0.0019, respectively). Each bar represents average ± s.e.m.
Figure 3Flow cytometric analysis of lymph node γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse or experiment and line the median. Representative dot plots showing IL-17A (A) or IL-17F (D) and IFNγ production in γδ T cells before (steady-state) and after IMQ-induced psoriasis. Numbers in gate indicate % positive cells; numbers outside the gate indicate mean fluorescence intensity of IL-17A or IL-17F. (B, E) Frequency of IL-17A+ (B) and IL-17F+ (E) γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. (C, F) Quantification of mean fluorescence intensity (MFI) of IL-17A (C) and IL-17F (F) staining in γδ T cells after IMQ-induced psoriasis (each color represents a different experiment). Frequency of IL-17A+ and IL-17F+ γδ T cells or IL-17A (G) and IL-17F (H) MFI following culture with IL-1β, IL-23 or nothing. Each symbol represents one experiment.
Loss of Snai1 predisposes differentiation towards secretory enteroendocrine and Paneth cell fate in small intestinal epitheliumTo analyse the effect of Snai1 knockout in the mouse small intestine, AhCre Snai1+/+ (control) and AhCre Snai1fl/del (test) adult mice were injected with β‐NF on four consecutive days to induce Cre‐mediated recombination.Figure 2To analyse the effect of Snai1 knockout in the mouse small intestine, AhCre Snai1+/+ (control) and AhCre Snai1fl/del (test) adult mice were injected with β‐NF on four consecutive days to induce Cre‐mediated recombination. Tissue was harvested for analysis on day 5. Periodic Acid Schiff (PAS) staining was used to detect goblet cells in sections of small intestine from control and Snai1 KO mice. Graph shows number of goblet cells per crypt-villus axes (n = 3).Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells (marked by arrows). Quantification of enteroendocrine cells per crypt-villus axes in control and Snai1 KO tissue revealed a significant increase in the number of enteroendocrine cells following Snai1 loss (n = 3, P = 0.004).Immunohistochemistry for lysozyme was used to detect Paneth cells. Quantification of Paneth cells in control and Snai1 KO indicates a significant increase in Paneth cell numbers following knockout of Snai1 (n = 4, P = 0.002). Measurement of villus length (μm) (n = 3, P = 0.013) and quantification of enterocytes per crypt-villus unit demonstrate a significant reduction in absorptive enterocytes following Snai1 loss (n = 3, P = 0.036).Measurement of villus length (μm) (n = 3, P = 0.003) 8 days after β‐NF induction demonstrates a significant shortening of villi. Bars represent mean ± SD. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 20 μm.
Figure 4(A Results of CRISPR-Cas9 screening in combination with AZD8186 100nM (A) The dot-plots show for each gene knocked-out by sgRNAs the fold change (log2) between treated conditions (AZD8186 and vehicle in the fluorescence signal (anti-phosphoS6 immunofluorescence) versus the p-value of the difference calculated by two sided t-test. The plots represent means of biological triplicates. Genes for which it was calculated a p<0.0001 and Log2 (fold change)>0.25 are reported and highlighted in green; genes having a 0.0001 0.25 are reported and shown in red. Gefitinib combined with AZD8186 represents the positive control of the experiment (A). <0.05> B) Results of CRISPR-Cas9 screening in combination with GDC0941 400nM (B). The dot-plots show for each gene knocked-out by sgRNAs the fold change (log2) between treated conditions GDC0941, and vehicle in the fluorescence signal (anti-phosphoS6 immunofluorescence) versus the p-value of the difference calculated by two sided t-test. The plots represent means of biological triplicates. Genes for which it was calculated a p<0.0001 and Log2 (fold change)>0.25 are reported and highlighted in green; genes having a 0.0001 0.25 are reported and shown in red. <0.05> (C) Box & Whisker plot showing the fold change in fluorescence signal between vehicle and GDC0941-treated conditions for MDA-MB-468 cells transduced with non-target Control, GNB2 or GNG5 sgRNAs (N=2 or 3). Data presented in a box and whisker plot with the central band indicating the median, the upper and lower extremes of the box or hinge being the third and first quartiles respectively and the whiskers extending to the most extreme data values within 1.5 times the inter-quartile range. Statistical significance of unpaired t-test ****P<0.0001. (D) Biochemical analysis of GNB2 KO cells. Cell lysates of MDA-MB-468 parental cells and three MDA-MB-468 GNB2 KO clones were probed with the indicated antibodies. Quantification of the bands was performed by ImageLite software.
Figure 3The regulation of metabolic pathways was reconstructed based on the metabolomic data of latently infected Jurkat T-cells (2D10) as compared to their uninfected counterpart (E6). Enzymes are shown in italics. The figure includes the main energetic pathways described in the paper, as well as relevant connections with pathways that were significantly enriched in the analysis shown in Figure 2A (grey boxes). The red arrows indicate the proposed path of the glucose carbon chains in latently infected cells. Solid lines indicate direct connections. Dashed lines indicate indirect connections involving intermediate metabolites not shown in the figure. Networks were built using the Cytoscape software (http://www.cytoscape.org) and the Metscape plugin (http://metscape.ncibi.org/tryplugin.html) (Gao et al, 2010) and adapted using Adobe Illustrator (v 16.03). ACLY = ATP citrate lyase; ACO = aconitase; ADPGK = ADP dependent glucokinase; ALDO = fructose 1,6 bisphosphate aldolase; ENO1 = enolase 1; FBP1 = fructose-bisphosphatase; PFK = phosphofructokinase 1; FH = fumarate hydratase; H6PD = hexose-6-phosphate dehydrogenase; HK1 = hexokinase 1; G6PD = glucose-6-phosphate dehydrogenase; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; GCK = glucokinase; GPDH = glycerol 3-phosphate dehydrogenase; GPI = glucose-6-phosphate isomerase; KORA = ketoglutarate dehydrogenase A; IDH = isocitrate dehydrogenase; LDHAL6A = lactate dehydrogenase A like 6A; MDH1 = malate dehydrogenase 1; PCK1 phosphoenolpyruvate carboxykinase 1; PFK = phosphofructokinase; PGAM4 = phosphoglycerate mutase family member 4; PGD = 6-phosphogluconate dehydrogenase; PGK1 = phosphoglycerate kinase 1; PGLS = 6-phosphogluconolactonase; PGM1 = phosphoglucomutase 1; PKLR = pyruvate kinase L/R; PRPS1 = ribose phosphate pyrophosphokinase1; RPIA = ribose 5-phosphate isomerase; SDHA = succinate dehydrogenase complex flavoprotein subunit A; SUCD = succinate semialdehyde dehydrogenase; TKT = transketolase; TPI = triose phosphate isomerase.
Atg36 localises to peroxisomes and induces their autophagic degradation.Figure 3(A) N‐ (left) and C‐ (right) GFP‐tagged ATG36 under control of the GAL1 promoter was induced in atg36Δ or atg1Δ cells. Early (2 h) and later (5 h) time points after induction are shown. Pexophagy is assessed using the peroxisomal marker HcRed-PTS1 expressed from the constitutive HIS3 promoter. (B) Pex11-GFP western blot showing pexophagy in WT cells grown 6 h in galactose medium (0) then switched to starvation medium for 1, 2 or 3 h as indicated. (W), WT (empty plasmid); (N), GAL1-mRFP-ATG36; (C), GAL1-ATG36-mRFP; (U), GAL1-ATG36. WT shows pexophagy by endogenous Atg36. (C) Pex11-GFP pexophagy of WT cells containing either empty plasmid (WT) or GAL1-mRFP-ATG36 grown 18 h in oleate medium (0) then switched to oleate medium containing 2% galactose and harvested at 3, 6 or 22 h as indicated.
Viral loads in tissues collected from rhesus macaques infected with SARS-CoV-2.Figure 6. Viral loads in tissues collected from rhesus macaques infected with SARS-CoV-2.Four adult rhesus macaques were inoculated with SARS-CoV-2 isolate nCoV-WA1-2020 and euthanized on 3 dpi. Thirty-seven tissues were collected at necropsy and analyzed for the presence of viral RNA by qRT-PCR. Tissues are grouped by lung lobes (a), with red symbols indicating tissues from which virus could be isolated in Vero E6 cells; other tissues from the respiratory tract (b); lymphoid tissues (c); gastrointestinal tissues (d); the central nervous system (e) and remaining tissues (f). Blue symbols in b-e indicate that viral mRNA was also detected in these tissues. Identical symbols have been used to denote identical animals throughout the figures in this manuscript. R: right; L: left; LN: lymph node.
Figure 2(A) Kymograph of CohesinHalo488 in the presence or absence of Wapl-Pds5. CohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with or without Wapl-Pds5, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. Scale bar, 1 μm.(B) MSD versus time of cohesin particles in (A) are shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.). (C) Acetylated- or unacetylated-cohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with Wapl-Pds5, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.). (D) Acetylated cohesinHalo488 was treated as in (C), the DNA-bound cohesin was further incubated with buffer, Wapl-Pds5, or Wapl-Pds5 plus Sororin, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).