EMBO/sd-character-level-ner huggingface dataset:
Genes with distal major PASs have higher gene expression levels and sequence conservation than those with proximal ones.Figure 2Sequence conservation comparison of the core PAS regulatory regions between major PASs with different locations. Upstream: upstream of last stop codon, 3' UTR(S): the only PAS in the 3' UTR, 3' UTR(F): the most proximal PAS in the 3' UTR, 3' UTR(M): the middle PAS in the 3' UTR, 3' UTR(L): the most distal PAS in the 3' UTR. (Mann-Whitney U test, *P < 0.05; **P < 0.01; ***P < 0.001). Genes with major PAS located in 3' UTR have on average higher major PAS usage (B) and gene expression level (C) compared to genes with upstream major PASs (Mann-Whitney U test, ***P < 0.001). dN/dS ratio comparison between genes with different major PAS locations (Mann-Whitney U test, *P < 0.05; **P < 0.01; ***P < 0.001). Among genes with exactly three PASs in 3' UTR, genes with major PAS located proximally have lower sequence conservation in the core regulatory regions of the major PASs and are under weaker selective constraint in their coding sequence Among genes with exactly three PASs in 3' UTR, genes with major PAS located proximally have lower sequence conservation in the core regulatory regions of the major PASs compared to the other genes. (Mann-Whitney U test, *P < 0.05; **P < 0.01; ***P < 0.001).
Figure 2representative SeaHorse time courses of OCR and ECAR respectively; where are indicated the following compounds added: oligomycin (olig.), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), rotenone plus antimycin A (R/A), glucose (Glu), 2-deoxyglucose (2DG). Each point in the OCR and ECAR time-courses is the average ± S.D. of four technical replicates. OCR/ECAR ratio; (4 technical replicates each); Basal, (resting OCR)/(ECAR +Glu); Max, (OCR+FCCP)/(ECAR+Olig.) Cellular NAD+ and NADH content and NAD+/NADH. Confocal microscopy analysis of the mitochondrial membrane potential DYm. Pictures on the right, representative imaging of cells loaded with tetramethylrhodamine, ethyl ester (TMRE); histogram on the left, averaged fluorescence intensity/cell ± SD of n=3 independent biological replicates; *, P < 0.05.
Figure 6(A) Representative images of control (upper panel) and α-synuclein loaded neurons (middle, 0.5µM, and bottom, 1µM, panels) after 16 h. In red: α-synuclein fibrils, in green: MAP-2 and in blue: nuclei. Scale bar represents 10 μm. (B) Representative images of donor (upper panels) and acceptor neurons (bottom panels) after 72 hours in co-culture. Donor neurons were loaded with α-synuclein fibrils prior to co-culture with CTG-labelled acceptor neurons and then cells were labelled either with MAP-2 (first and third panels) or Lamp1 (second and fourth panels). In red: α-synuclein fibrils, in green: CTG, in white: MAP-2/ Lamp1 and in blue: nuclei. Scale bar represents 5 μm.(C) The graph bar shows the percentage of donor (white bar) and acceptor (black bar) neurons containing α-synuclein puncta. Data show mean ± s.e.m.(D) The box plot depicts the number of puncta in donor (white) and acceptor (grey) neurons.(E) Box plot showing the distribution of the average size of α-synuclein puncta in donor (white) and acceptor (grey) neurons.(F) The graph bar represents the percentage of α-synuclein puncta co-localized with lysosomes in donor (white) and acceptor (black) neurons. Data show mean ± s.e.m. Graphs in C, D, E and F correspond to data from 3 independent experiments analysed after 72 hours of co-culture (****, p < 0.0001 compared to donor by two-tailed Mann Whitney test).
TBL1-depletion leads to reduction in PI3 kinase p110α and cell cycle-associated proteins.Figure 5TBL1-depletion leads to reduction in PI3 kinase p110α and cell cycle-associated proteinsPanc02 cells with stable expression of shRNA were implanted subcutaneously into C57Bl6/Nmice. Seven days later, mice were treated with 20 mg/kg gemcitabine delivered by intraperitoneal injection. After 21 days, mice were sacrificed, tumors were removed and proteins were extracted and immunoblotted.
Relevance of the Ctf19-Mcm21 binding motif for cell viability.Figure 8A) Representative images of dilution-series growth assay of haploid S. cerevisiae clones with cnn1Δ, Okp1_fl, Okp1_cmΔ, Okp1_fl cnn1Δ, or Okp1_cmΔ cnn1Δ, grown for two or three days on solid YPD agar or on solid YPD agar with 20 μg ml−1 benomyl. We show two unique clones each for Okp1_fl cnn1Δ or Okp1_cmΔ cnn1Δ. Arrow indicates decreasing cell-density. Spots are from cell samples that we sequentially diluted four fold. B) Representative images of haploid S. cerevisiae spores from dissection of three tetrads (we placed the four spores from each tetrad row-wise) from heterozygous diploid cells with Okp1_fl mad1Δ or Okp1_cmΔ mad1Δ grown for four and three days, respectively on solid YPD agar at 25 °C. Coloured circles indicate spore genotypes (see bottom). C) Representative images of haploid S. cerevisiae with mad1Δ, Okp1_fl mad1Δ, or Okp1_cmΔ mad1Δ; native: S. cerevisiae S288C type (identifier DDY904 in Table EV6). Dilution is as we describe for A. We show two unique clones for Okp1_fl mad1Δ. D) Quantification of spore viability after meiosis, of spores of dissected tetrads from homozygous diploid S. cerevisiae clones with either Ctf19_1-954Δ (without Ctf19 basepairs 1-954; leaving the 3' end of Ctf19, and, on the complementary DNA strand, the IRC15 gene, which encodes a microtubule binding protein (Keyes & Burke, 2009) intact), Ctf19Δ (that lacks the entire Ctf19), Okp1_fl, Okp1_cmΔ, or Okp1_cmΔnnΔ. Quantification is from spores from multiple tetrad dissections.
Pharmacological PI3Kα inhibition prevents the rapid progression of cfDNA-positive PDAC.Figure 6A, KPC mice diagnosed with an aggressive carcinoma were given daily oral doses of BYL-719 (50mg/kg) or of vehicule (0.5% methyl cellulose with 0.2% Tween-80). n = 6 in each group B, The protein levels of pAkt (Ser473), total Akt, pERK (Thr202 and Tyr204) and total ERK were observed by western blot in spleen, lung and pancreas tissue lysates after treatment with oral doses of vehicle (0.5% methyl cellulose with 0.2% Tween-80) or of BYL-719 (50mg/kg). C, Quantification of tumour volume (expressed in fold change) D,E, quantification of micro- and macro-metastatic foci and the relative metastasis area (comprising metastases in the spleen, liver and lung) of KPC mice treated with oral doses of vehicle (0.5% methyl cellulose with 0.2% Tween-80) or of BYL-719 (50mg/kg). F, Development of ascites in KPC mice after treatment with oral doses of vehicle (0.5% methyl cellulose with 0.2% Tween-80) or of BYL-719 (50mg/kg). G, Survival curve of KPC mice treated with the vehicle or BYL-719. Log rank test, * p<0.05. H-J H, Representative images and i, quantification of Ki67 positive cells in pancreatic tumours and in J, metastasized liver sections after treatment with oral doses of vehicle (0.5% methyl cellulose with 0.2% Tween-80) or of BYL-719 (50mg/kg). K-L K, Treatment regimen of the tail vein injection experiment in nude mice treated with the PI3Kα inhibitor or vehicle (n is indicated), quantification at two time points and L, representative images of luminescence via IVIS® Spectrum in vivo imaging system. M, Quantification and representative CK19 IHC of R211-luc lung micro-metastasis from vehicle or BYL-719 treated mice. n=6 in vehicle; n=5 in BYL-719 group. N, Tail vein injection experiment in C57/B6 mice of murine syngeneic pancreatic cancer cells (Kras mutated A338, Kras mutated and half PI3Kα inactive, A260; n>8 in each group), quantification of micro- and macro-metastasis at final time point.
Both GABARAP/GATE‐16 and LC3 protein subfamilies are essential for autophagic flux.Figure 3(A) Bulk protein degradation was tested in HeLa cells that were transfected with non‐targeting siRNA (control siRNA), a pool of GABARAP/GATE‐16 siRNAs, or a pool of LC3 siRNAs. The rate of long‐lived protein degradation was measured in cells incubated in either αMEM (control) or EBSS (starvation) medium, in the presence or absence of 10 mM of 3‐MA, 72 h after siRNA transfection. Values expressing the protein degradation percentage are represented as the mean±s.d. of three determinations. (B) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (right panel) were transfected with a pool of GABARAP/GATE‐16 siRNAs or a pool of LC3, respectively, and with non‐targeting siRNA. Seventy‐two hours after transfection, the cells were collected after 5 h of starvation and the relative level of GFP‐LC3B or GFP‐GATE‐16 was measured using flow cytometry. Values represent the mean±s.d. of three experiments.
Figure 4A. HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of 20 μM pifithrin-α (Pft). Basal and phospho-active forms of ATM, CHK2, p53, and GSNOR were assessed by Western blot. Phospho:basal level ratios of ATM, CHK2 and p53 (normalized to Vinculin), along with densitometry of GSNOR immunoreactive bands (normalized to GAPDH) are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant. B. HEK293 cells were transfected with a pool of siRNA against p53 (sip53) or with control siRNA (siScr) for 24 h and treated for additional 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM and CHK2, as well as p53 and GSNOR were assessed by Western blot. Phospho:basal level ratios of ATM and CHK2 (normalized to Vinculin), along with densitometry of GSNOR immunoreactive bands (normalized to GAPDH) are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant. C. HCT116 cells expressing the wild type form of p53 (p53wt), or an empty vector (Empty, selected as negative control), were treated for 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM and CHK2, p53 and GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls. Phospho:basal level ratios of ATM and CHK2, along with densitometry of p53 and GSNOR immunoreactive bands are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant.
Figure 2(A) Tumor growth delay was measured in the different groups, as indicated (n=6 mice per group). Anti-PD-L1 was given at days 0 3, 6 and 9 (black arrows). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 is shown (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiment (B) Quantitative data on the percentage of gated cells are shown (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).
Exposure to CDK7i induces melanoma dedifferentiationA.Figure 2A. Volcano plots were used to demonstrate differentially expressed genes as determined by RNA-seq in either MM047CDK7i-R vs MM047 (top), MM074CDK7i-R vs MM074 (middle) or MM074BRAFi-R vs MM074 (bottom). Red dots show significantly over-(top) or under-(bottom) represented mRNA in drug-resistant cells compared to parental cells. All data were evaluated with the DESeq2 R package. The value for a given gene is the normalized gene expression value relative to the mean of all samples belonging to the same condition. B. Proportional Venn diagrams indicating the number of up- (left) and down- (right) regulated genes in MM047CDK7i-R and MM074CDK7i-R compared to the parental MM047 and MM074, respectively. The number of genes overlapping between the data sets is indicated. 261 genes were found up-regulated and 241 down-regulated in MM047CDK7i-R and MM074CDK7i-R. Hypergeometric p-value is indicated. C. Genes characterizing the melanocytic-type and mesenchymal-like transcription signatures (Widmer et al., 2012) have been plotted on a Heatmap and are shown in relation to their expression in different melanoma cells. RPKM values are represented as z-score. The group of genes related to pigmentation has been highlighted in red. The color key shows the log2 expression values. Yellow color stands for high expression and dark violet for low expression. D. Protein lysates from MM074, MM074BRAFi-R or MM074CDK7i-R were immuno-blotted for indicated proteins. Molecular sizes of the proteins are indicated (kDa). The numbers below the gel lanes represent relative protein level, which was determined from the band intensity using ImageJ software normalized to each relative Vinculin control.
Figure 1Cartoon structure of optimized, inhibited parkin from x-ray crystal structure (PDB 5N2W)(Kumar et al, 2017). The structure shows the Ubl (grey), RING0 (brown), RING1 (blue), IBR (green) and RING2(Rcat) (pale green) domains along with the tether region (purple). A schematic block diagram is shown above to illustrate the domains. The pUb (yellow) molecule is shown bound to the RING0/RING1 interface. Zn atoms are shown as spheres and regions of parkin not observed in the crystal structure are indicated as dashed lines. Selected examples of % deuterium uptake measured by HDX mass spectrometry to show differences between full-length autoinhibited parkin (black lines) and phosphorylated parkin (pParkin) in complex with pUb (orange lines ). Each plot shows the % deuterium uptake for the peptide indicated as a function of time. Differences in % deuterium uptake between auto-inhibited parkin and pParkin:pUb state are plotted at the one minute time point. The width of the bars shown represent the lengths of the peptides observed and measured while the heights represent the relative difference in hydrogen-deuterium exchange between the two species. Error bars represent standard deviation above the average for triplicate measurements. Positive bars indicate a greater deuterium uptake in the pParkin:pUb state compared to the autoinhibited state indicating greater exposure to solvent and/or weaker hydrogen bonding, while a negative change indicates the pParkin site is more protected. The domain structure of parkin is shown below the data to indicate the location of peptides. Relative HDX differences are mapped to the structure of parkin:pUb (PDB 5N2W) where the pUbl domain has been arbitrarily positioned away from the remainder of the protein. Regions where HDX were faster in the pParkin:pUb complex relative to parkin alone at the one minute time point are indicated in red, while those regions in blue exchange more slowly using the gradient shown. Several regions not visible in crystal structures were modelled into the structure using the Modeller tool (Eswar et al, 2006) in Chimera (Pettersen et al, 2004). The arrows at the top of the figure indicate the pUbl domain samples multiple conformations based on its overall faster HDX in the phosphorylated state and previous NMR and analytical sedimentation velocity experiments (Aguirre et al, 2017).
Figure 4A The immunoprecipitation analysis. The cytoplasmic ENTREP (wild or mut1+2 mutant) co-precipitated with EPN1myc in the presence of ITCH wild or C830A. The blot bands were semi-quantified using ImageJ software. ENTREP ratio was caluculated as a ratio of ENTREP in the myc-coprecipitaed sample to that of the total lysate. The ratios shown as mean ± SD from three biological replicates. P-values were obtained by Student's t-tests and P < 0.005 was considered as statistically significant. No significant difference was observed. B Representative images of the proximity ligation assay (PLA) using Cos7 cells transfected with indicated vectors. PLA signals, granular dots of green color, were presented with DAPI images. Scale bar, 10mm. C The box-and-whisker plot presentation of the PLA. A minimum of 10 cells per condition were counted from two independent experiments. P-values obtained by Student's t-tests and P < 0.05 was considered as statistically significant. * P < 0.05. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers. D The expression of endogenous ENTREP in HMEC. ENTREP co-localized with Clathrin, RAB5, RAB7 and RAB11. Inserts, the ring-shaped structures of the marked areas. The images are representative of at least two independent experiments. Scale bar 10mm. E Co-localization of ENTREP with ITCH and EPN1. Cos7 cells transfected with indicated vectors were stained with specific antibodies and observed under a laser confocal microscope. Arrowheads, full length ENTREP (wild or mut1+2 mutant) expression at the plasma membrane. Inserts, the high-magnification images of the marked area indicating the co-localizaion of ENTREP-FLAG, EPN1myc and Halo-ITCHwild. The images are representative of at least two independent experiments. Scale bar 10mm.
Figure 10A) We propose a sorting mechanism that redirects Ptc to the basal membrane for its presentation for Hh-reception in the cytonemes of receiving cells. In black numbers and arrows we show the vesicle-sorting route for Ptc presentation at the basal plasma membrane, 1) endocytosis of Ptc from apical membranes, 2) late endosome structures to form MVBs that 3) fuse to the basal plasma membranes exposing Ptc for Hh reception. In red numbers and arrows we show the parallel vesicle-sorting route directing Hh/Ptc degradation after 1) reception and 2) endocytosis at basal membranes, towards 3) MVB formation for degradation at lysosomes. This novel mechanism for polarized Ptc trafficking requires components of the ESCRT machinery (MVB-mediated processes such as recycling), the Tetraspanin proteins (to define MVB and EV membrane domains) and the SNARE proteins (to regulate vesicle fusion). Potential mechanisms for the transport within cytonemes of two vesicle types, for Ptc membrane deposition and post-reception, are not shown in the model as they are still unexplored. B) Schematic view of the effect over Hh signalling after transient inhibition of the MVB-mediated recycling and fusion process proposed for Ptc localization at basal membranes. Top panel shows Hh dispersion in a wild type situation, while bottom panel shows an extended dispersion after transient conditions that reduce Ptc basal membrane exposure and thus decrease Hh reception, flattening the signaling gradient.
ITSN2 deficiency lowers TCR activation threshold.Figure 5a t-SNE plot highlighting regulated pY sites (for sake of space, only the gene names are reported). Phospho-sites of STAM2 and ITSN2 are displayed in red. b Cas9-EGFP OT-I CD8+ T cells were transfected with control sgRNA (sgEGFP) or with two different sgRNA targeting Itsn2 (sgITSN2-1 or sgITSN2-2). Transfected cells were stimulated for 48 h with N4 peptide MHCc Proliferation of OT-I Cas9-EGFP CD8+ T cells transfected with EGFP or with ITSN2 sgRNA activated for 48 h with N4 peptide MHC tetramers (0.01-0.1 nM) in the presence or absence of soluble anti-CD28 antibody or with PMA and ionomycin (PMA/Iono) or with IL-7. Data are presented as mean ± standard deviation (SD) from four proliferative measures of two independent sgEGFP and sgITSN2 transfections. Data are representative of three independent experiments. d IFN-γ production of cells treated as in c was assessed by intracellular staining after 24 h of stimulation. e Proliferation of similar cells as in c activated for 48 h in vitro with a range of increasing doses of N4 peptide MHC tetramers in the presence of soluble anti-CD28 antibody.
Figure 2A Three pairs of small interfering RNA against TRAF3IP3 were transfected into HEK293T cells respectively and immunoblotting were then employed to examine the knockdown efficiency. A pair of scrambled RNA oligoes was transfected and signified as con. B Three pairs of si-TRAF3IP3 together with IFNβ-luciferase reporter were transfected into HEK 293T cells. Twenty-four hours after transfection, the cells were infected with (SeV) or without (mock) Sendai virus, which were harvested six hours post infection to measure the luciferase activity. C Si-TRAF3IP3 (oligo #1) were transfected into HEK 293 cells, which were then treated with mock, VSV, SeV, poly(I:C) or HSV-1. After various treatments, IFNB inductions were measured by qPCR. D Immunoblotting to detect TRAF3IP3 in wild type and Traf3ip3-/- HEK293T cells. E Wild type and Traf3ip3-/- HEK293T cells were treated with or without VSV, SeV or poly (I:C) as indicated for twelve hours. IFNB induction was measured by qPCR. F Wild type and Traf3ip3-/- HEK293T cells were infected with GFP-VSV. Fluorescent images were taken to examine GFP-VSV proliferation at eight hours post infection (left). Scale bar represents 10 micrometers. GFP-VSV titers were quantitated by plaque assay (right). G Various expression constructs were transfected into WT and Traf3ip3-/- HEK293T cells. Thirty-six hours after transfection, IFNB induction was measured by qPCR. H Mavs-/- HEK293T cells were transfected with empty vector, pcDNA-FLAG-MAVS, pcDNA-FLAG-MAVS-(Region III only), or pcDNA-FLAG-MAVS-(ΔRegion III), together with pcDNA3-FLAG-TRAF3IP3 or not as indicated. Twenty-four hours after transfection, cells were infected with SeV for twelve hours. IFNB induction was measured by qPCR.
a) Ingenuity analysis identifies Hif1α as a key upstream regulator (activation z-score=2.f9(a) Ingenuity analysis identifies Hif1α as a key upstream regulator (activation z-score=2.003; P=9.43e−08) that connects to a set of upregulated (red node) or downregulated (green nodes) genes in isolated KRas;Atg5fl/fl, non-transformed pneumocytes 48 h after infection with AdCre. Expression was determined by RNAseq. Orange edge, leads to activation; yellow edge, inconsistent state; grey edge, effect not predicted. (b) Protein expression of Hif1α, Atg5 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in isolated KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes (analysed at 4 days after AdCre infection) and tumours purified from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice at week 2 after AdCre inhalation. Quantifications of the hypoxia-inducible factor (HIF) 1α/GAPDH ratios are shown for each lane; two independent blots are quantified. (c) CD39, Atg5 and as a control β-actin protein expression in isolated Atg5fl/+, Atg5fl/fl, KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes. Protein lysated were analysed at 4 days after AdCre infections by western blot. Quantifications of the CD39/β-actin ratios are shown for each lane, normalized to the Atg5fl/+ samples. Two independent blots are quantified. (d,e) CD39, Atg5 and as a control β-actin protein expression in isolated KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes treated with the (d) HIF1α inhibitor 3-(2-(4-adamantan-1-yl-phenoxy)-acetylamino)-4-hydroxybenzoic acid methyl ester or left untreated and (e) the Nrf2 inhibitor Brusatol (100 nM) or the ROS scavenger N-acetyl-L-cysteine (5 mM). Protein lysates were analysed on day 4 by western blot. Quantifications of the CD39/β-actin ratios are shown for each lane. The inhibitors were present during the entire 4 day culture period.
Figure 7Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread cultured wild-type control (WT Control), and okadaic acid treated wild-type (WT + OA) and Smc1β-/- (Smc1β-/- + OA) spermatocytes.A-B WT control cultured spermatocytes in (A) late zygotene (L. zyg.) and (B) metaphase I (M I). Arrows indicate the cell poles.C-D WT cultured spermatocytes treated with okadaic acid in (C) late zygotene (L. zyg.) and (D) metaphase I-like (M I-like).E-F Smc1β-/- cultured spermatocytes treated with okadaic acid in (E) pachytene-like (Pac.-like) and (F) metaphase I-like (M I-like). Arrowheads indicate nucleolar-associated round bodies. The unsynapsed axial elements of the sex chromosomes (X,Y) in prophase I, as well as the sex bivalent (XY) in metaphases I are indicated. Scale bar: 10 µm.