EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) Schemes of the CDK threshold sensors based on Sic1 used in panels 'C' and 'D'. The sensors contain the non-inhibitory domain of Sic1 (1-215), have mutated RxL and LP docking sites and insertion of 4 residues between T33 and T45. The docking sites are inserted to the position of Sic1 RxL motif. (B) Alignment of the Sic1 segment with the different introduced docking motifs. (C) Plot showing the mean±SEM fluorescence intensities of the Sic1-based threshold sensors shown in 'A' containing the indicated cyclin docking motif. (D) The time from Start to the fluorescence levels of the threshold sensors with indicated docking motifs decreasing to 50% of peak level was measured in single cells. The black lines show median±95% CI. The numbers above the plot show median degradation timings of the indicated sensors. (E) In vitro phosphorylation kinetics of Sic1(T33+16T45 rxl llpp ΔC) with the indicated cyclin docking motif by Clb5-Cdk1. The data is from two replicate experiments. (F) Analysis of multisite phosphorylation of Sic1(T33+16T45 rxl llpp ΔC) with different docking motifs using Phos-tag SDS-PAGE autoradiography. The experiment was performed twice, a representative example is shown. (G) The fraction of maximally phosphorylated form from total phosphorylation signal was quantified from Phos-tag gels as shown in panel 'F'.
Involvement of Atg14L in the initial phase of autophagosome formation.Figure 1.(a) HEK293 cells stably expressing GFP-DFCP1 were transfected with or without shRNA against Atg14L. The cell lysates were subjected to immunoblotting with the indicated antibodies. (b) GFP signals were observed in these cells before or after starvation. (c) The number of GFP-positive puncta per cell was counted; mean ± SD values are presented. N, nutrient rich; S, starvation. (d) HEK293 cells were lysed and subjected to subcellular fractionation as described in Materials and methods. Equivalent amounts of total, cytosolic, and ER fractions were subjected to immunoblotting with each organelle marker antibody. The percentage of recovery in each fraction is presented as mean ± SD values. (e) HEK293 cells stably expressing GFP-DFCP1 were transiently transformed with Flag-tagged Atg14L and subjected to starvation. The cells were fixed and stained with the indicated antibodies. Insets show higher magnification of the boxed areas. (f) HEK293 cells were transfected with GFP-Atg14L with or without mCherry-linked dominant-negative ULK1, and these cells were cultured in complete or starvation medium for 60 min. Bars, 10 µm.
cGAMP loading of VLPs enhances induction of CD4 Tfh responses.Figure 4Immunisation with cGAMP-VLPs enhances accumulation of Tfh cells in the draining lymph node. T follicular (Tf) cells were identified by flow cytometry as CD4+CD44+CXCR5hiPD1hi cells and were further subdivided into Tfr cells (FoxP3+) and Tfh cells (FoxP3-). The gating strategy is shown in (A) Immunisation with cGAMP-VLPs enhances accumulation of Tfh cells in the draining lymph node. T follicular (Tf) cells were identified by flow cytometry as CD4+CD44+CXCR5hiPD1hi cells and were further subdivided into Tfr cells (FoxP3+) and Tfh cells (FoxP3-). the percentages of Tf, Tfh and Tfr cells within CD4+ cells are shown in (B). Immunisation with cGAMP-VLPs enhances accumulation of Tfh cells in the draining lymph node. T follicular (Tf) cells were identified by flow cytometry as CD4+CD44+CXCR5hiPD1hi cells and were further subdivided into Tfr cells (FoxP3+) and Tfh cells (FoxP3-). The ratio of Tfh/Tfr is shown in C. Immunisation with VLPs induces germinal centre formation. Germinal centre B cells were identified by flow cytometry as B220+IgD-CD95+GL7+ cells. The gating strategy is shown in (D) Immunisation with VLPs induces germinal centre formation. Germinal centre B cells were identified by flow cytometry as B220+IgD-CD95+GL7+ cells. the percentage of germinal centre B cells amongst B220+ cells is shown in (E). F. Immunisation with cGAMP-VLPs increases production of antibody-secreting cells. Cells from draining lymph nodes and spleens were seeded in ELISPOT plates coated with cGAMP-VLP lysates. After overnight incubation, cells producing VLP-specific IgG antibodies were identified using an anti-IgG Fc antibody.
Figure 5Our work shows that virtually all overlap microtubule bundles containing PRC1 are linked to sister k-fibers during metaphase. Likewise, each pair of sister kinetochores is associated with a single overlap bundle, which connects their k-fibers as a bridge. Microtubules are represented in green, kinetochores in magenta and PRC1 cross-linkers as black crosses.
a) Representative electron microscopy images of autolysosomes (left panels), autophagosomes (middle panels) and mitophagy (right panels), all indicated by arrows, in lung tumour cells from control KRas;Atg5fl/+ mice 18 weeks after AdCre inhalation.f2(a) Representative electron microscopy images of autolysosomes (left panels), autophagosomes (middle panels) and mitophagy (right panels), all indicated by arrows, in lungtumour cells from control KRas;Atg5fl/+ mice18 weeks after AdCre inhalation. Note the near-to-complete absence of such structures in the lungtumour cells from KRas;Atg5fl/fl littermates. Very rarely, we found cells with one autolysosome (left panels) or one single autophagosome (middle panels; arrows) in KRas;Atg5fl/fl tumours, whereas such structures can frequently be seen within single KRas;Atg5fl/+ tumour cells. Of note, the presence of a single autophagosome or a single autolysosome was scored as a positive event in Fig. 1e. Also note aberrant mitochondrial morphologies and increased mitochondrial numbers in KRas;Atg5fl/fl tumour cells. * indicates lamellar bodies. Scale bars, 2 μm. (b) Mean numbers (±s.e.m.) of mitochondria per lungtumour cell from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice18 weeks after tumour induction. Numbers of mitochondria were determined by electron microscopy. n≥8 mice per genotype and a minimum of 20 intact cells were counted per sample. ***P0.001 (χ2-test assessing the genotype effect in a generalized linear model with log link). (c) Mitochondria (arrows) in KRas;Atg5fl/+ and KRas;Atg5fl/fl lung tumour cells 18 weeks after AdCre inhalation. Note markedly altered mitochondrial morphology, that is, disorganized matrix structures, rounding and enlargement in the autophagy-defective KRas;Atg5fl/fl cells. Au, autophagosomes; Au-L, autolysosome; *, lamellar bodies. Scale bars, 2 μm. (d-g) Bioenergetics profiling of purified tumour cells from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice (e) 6 weeks and (g) 18 weeks after tumour induction. Representative experimental data are shown. Oxygen consumption rates (OCR) were used to detect the bioenergetics profiles of purified tumour cells using a Seahorse Bioscience 24XF extracellular flux analyser. OCR±s.e.m. were recorded to construct bioenergetics profiles for basal respiration (BR), ATP turnover (AT), respiratory capacity (RC), spare respiratory capacity (SRC) and proton leakage (PL). A minimum of seven different samples were analysed for each group. *P0.05; **P0.01(unpaired, two-sided t-test).
Fig.Figure 6(A) Schematic depiction of the S6 protein (red) in the context of bacterial ribosome (after PDB:6WD0, left) and yeast mitochondrial ribosome (after PDB:5MRC, right) highlighting the position of the N-terminus of S6 (red arrow, only shown on the left image). Proteins homologous between bacterial and yeast structures are shown in orange, mitochondria-specific protein mS26 in teal and rRNA in grey. (B) On the right: MRP N-termini structural environment (solvent exposure): average number of atoms belonging to the same protein, other protein chains or RNA in the 13Å radius from the first structured amino acid Cα atom, plotted separately for proteins with their most N-terminus appearing in the structure vs proteins with precisely cleaved MTS, and other proteins which have a longer unstructured extension (see the classification legend on the bottom) calculated based on PDB:5MRC; on the right: same as on the left but only coordination number (number of protein Cα atoms in the 13Å radius (Hamelryck, 2005)) is calculated, ** p-value<0.01 (Mann-Whitney test), absolute values were measured without replicates; on the bottom: MRPs were divided in three groups depending on relative position of the MTS and structured residues: "No MTS (str)" - structure starts at the very N-terminus without a cleavable MTS, "Precise MTS" - MTS is cleaved off 1-2 amino acids before the structure starts, "Other" - unstructured uncleaved amino acids present before the structure start. (C) OXPHOS complex components usually have their N-termini free to develop an MTS (left). In contrast, mitoribosomes often employ the MRP N-termini in binding interfaces which puts structural constraints on the development of N-terminal MTS and promotes the evolution of internal targeting sequences that can also take part in ribosome assembly (right).
Figure 5A) Acetylation tracks for H3K9/14ac and H3K27ac ChIP-seq (on the left) and corresponding FPKM values by RNA-seq (on the right) for representative genes in YC, YT, OC and OT FAPs. B) Graphs showing RNA levels for the representative genes shown in (A) measured by qPCR. C) Graphs showing the H3K9/14 acetylation levels for the representative genes shown in (A) measured by ChIP-qPCR. D) Representative dot-plots of the flow cytometry analysis of KI-67 PE-Cy7 to monitor proliferating YC, YT, OC and OT FAPs. E) Boxplot showing the percentage of KI-67+ FAPs analysed in (D). F) Boxplot showing the total number of FAPs in the same experimental conditions described in (D). G) Representative dot-plots showing the flow cytometry analysis of FAP migration assay. YC, YT, OC and OT FAPs were labelled with PKH67-488 and analysed in the proximal (top panel) and distal (bottom panel) sections from the injection site in YC mice. H) Stacked bar chart showing the relative percentage of PKH67-488+ FAPs detected in the proximal (blue) and the distal (red) sections for the same experimental conditions described in (G). I) Histogram showing the level of glycolysis measured by Seahorse as extracellular acidification rate (ECAR) after glucose administration to YC, YT, OC and OT FAPs.
Accelerated aging phenotype in SirT7-/- mice.Figure 2A Representative 3D reconstructed CT scans showing increased kyphosis in 16 month old SirT7-/- mice compared with WT. B Representative gonadal fat pads from WT and SirT7-/- 16 month old mice. C Quantitation of body weight (left) and gonadal fat pad mass normalized to total body weight (right) (mean ± SEM; 4 samples per genotype). D IGF-1 protein levels in serum measured by ELISA (mean ± SEM; 3-8 mice per genotype and age group). E, F Dot plots of Lin-Sca1+cKit+ (LSK) cells (middle and right) from WT and SirT7-/- bone marrow cells. Cells were gated for negative staining of lineage markers B220, CD3, CD11b, CD19, Gr-1, and Ter-119 (Left), and analyzed for Sca1 and cKit expression (middle and right). (F) Quantitation of (E) (mean ± SEM; 4 mice per genotype). G Bone marrow, thymus, and spleen cell number in young WT and SirT7-/- mice (mean ± SEM; 3-5 mice per genotype). H mRNA expression of p16 gene normalized to GAPDH measured by RT-PCR from young and old WT and SirT7-/- fibroblasts (mean ± SEM; 3 samples per genotype). I, J Senescence-associated β-galactosidasestaining (I, blue) in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and grown for 7 days in selection media. (J) Quantitation of the number of senescent cells shown in (I) (mean ± SEM; 3 independent cell lines per genotype).*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by ANOVA Single Factor.
Distribution of selected amino acids in gliomas.Figure 4A) MALDI imaging of key metabolites in IDHwt (P3) and IDHm (E478) glioma PDX, compared to control brain (CB) sections. B) Quantification of metabolite distribution by MSI in 6 PDX with IDH1wt and IDH1m status (n=3 / group) and in contralateral control brain (CB). No significant differences between IDH groups were detected, although significant differences are found between tumors and control brain (glutamate, apsartate). Color code as in Fig. 1, NAA: N-acetylaspartic acid; NAAG: N-acetylaspartylglutamic acid.
Figure 1(A) Proliferation analysis of hematopoietic CD34+ precursors for up to 6 days (section i). (A) Neutrophil morphologic differentiation were assessed at day 6 (section ii). Controls were without treatment. White arrows indicate neutrophilnuclear segmentation. A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve. (A) Neutrophil bacterial killing were assessed at day 6 (section iii). Controls were without treatment. . A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve. (A) Neutrophil ROS production were assessed at day 6 (iv). Controls were without treatment. A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve. (B) qRT-PCR assessed RA-target gene expression after culturing CD34+ cells for 1, 2, and 6 days. (C) Flow cytometry analysis of CD66-CD18 co-expression after culturing CD34+ cells for 6 days, using anti-human CD66-PE and CD18-FITC antibodies (section i). Isotypes were used for controls (section i, top panel). CD66-CD18 co-expression was quantified in section ii. (D) Fresh peripheral blood (PB) collected from normal human donor. Bacterial killing was assessed in the presence or absence of PBneutrophils (section i). (D) Fresh peripheral blood (PB) collected from normal human donor. Bactericidal activities of neutrophils induced by Am80-GCSF from PB mononuclear cells were evaluated by ROS production (section ii). (D) Fresh peripheral blood (PB) collected from normal human donor. Bactericidal activities of neutrophils induced by Am80-GCSF from PB mononuclear cells were evaluated by bacterial killing assays (section iii). (E) Fresh PBneutrophils collected from normal human donor were tested for ROS production in the presence or absence of specific antibodies. Controls were with or without IgG. Normal IgG, φ; Ab, antibodies; Comb Ab, combined anti-CD18 and -CD11b antibodies. (F) Fresh PB mononuclear cells collected from normal human donors were treated for 3 days. Bacterial killing (sections i) and neutralization of bacterial killing in the presence of IgG or anti-CD18 antibody were assessed at day 3 (section ii).
Figure 3(A) WT or Clec4F-DTR+ C57BL/6 mice were treated i.v. with DT prior to s.c. inoculation in the left rear footpad with 103 PFU of CHIKV. Infectious virus was quantified at 24 hpi by FFA. Mean ± SEM. Data are pooled from two experiments, n=8. Mann-Whitney test; P > 0.05. (B) WT C57BL/6 mice were treated i.v. with PLL or CLL 42 h prior to s.c. inoculation in the left rear footpad with 103 PFU of CHIKV. Infectious virus at 24 hpi was quantified by FFA. Mean ± SD. Data are pooled from two experiments, n=8. Mann-Whitney test; ***P < 0.001.
Figure 1RNA-seq of liver 10h post Sham or CLP. Mice (n = 3/group) underwent a sham or CLP operation and were injected with GW7647 (10 µg/g) 6h post-surgery, after 4h (total of 10h), livers were isolated and RNA was prepared. Scatter plot showing log fold change (LFC) of all GW7647-upregulated genes (LFC > 0.8 and P < 0.05) in sham versus their LFC 10h after CLP. The red line represents the diagonal, the black line represents the real slope (0.3115) of the data. RNA-seq of liver 10h post Sham or CLP. Mice (n = 3/group) underwent a sham or CLP operation and were injected with GW7647 (10 µg/g) 6h post-surgery, after 4h (total of 10h), livers were isolated and RNA was prepared. Venn diagram depicting the amounts of genes upregulated (up) or downregulated (dn) by GW7647 in sham and CLP mice (LFC > 0.8 or < -0.8 and P < 0.05). RNA-seq of liver 10h post Sham or CLP. Mice (n = 3/group) underwent a sham or CLP operation and were injected with GW7647 (10 µg/g) 6h post-surgery, after 4h (total of 10h), livers were isolated and RNA was prepared. Top enriched gene ontology (GO) terms for genes that are downregulated in CLP mice without stimulation compared to unstimulated sham controls (LFC < -0.8 and P < 0.05). Composite of 3 datasets; CLP1 (6h after CLP), CLP2 (8h after CLP) and CLP3 (10h after CLP). Analysis was performed with the Enrichr tool. RNA-seq of liver 10h post Sham or CLP. Mice (n = 3/group) underwent a sham or CLP operation and were injected with GW7647 (10 µg/g) 6h post-surgery, after 4h (total of 10h), livers were isolated and RNA was prepared. Heatmap of differentially expressed genes in sham mice after GW7647 treatment, involved in β-oxidation of fatty acids (unit scale bar = log2 of the normalized counts). Confirmation of RNA-seq data via qPCR on pure hepatocytes isolated via flow cytometry-based sorting (n = 3/group). (E) Ppara and (F) Hmgcs2 mRNA expression is shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated using 2-way ANOVA analysis.
Figure 4Body weight gain over a 24-week period of high-fat feeding (n = 13-14 animals per group). Panel shows experimental design for Figures 4-6. Average daily food intake in g (n = 6 cages per genotype). Feed efficiency to evaluate weight gain per ingested J (n = 6 cages per group). Fecal energy loss over a 3-day period determined by combustion of lyophilized feces of a subgroup of HFD-fed mice (n = 9 animals per group). Oxygen consumption over a 90-hour period. Grays columns indicate dark phase (n = 6-8 mice per group). Average oxygen consumption specified for light phase, dark phase, and total (n = 6-8 animals per genotype). Total oxygen consumption plotted against body weight indicating that lower energy expenditure in GRBATKO animals is independent of body weight (n = 6-8 animals per group). Locomotor activity over a 90-hour period (n = 6-8 animals per group). Cumulative food intake over the 90-hour period specified for each phase and as grand total (n = 6-8 animals per group).
Figure 6Venn diagram showing the uc.291 up-regulated genes in si-uc.291 keratinocytes in common with the si-ACTL6A down-regulated genes. The 39 common genes are listed below and the EDC genes are indicated. The expression of the genes is inversely correlated. The inverse correlation among them functionally support the negative regulation of uc.291 in controlling ACTL6A functions. Schematic model of uc.291 role in regulating epidermal differentiation.
UBE2QL1 abrogates recruitment of VCP/p97, reduces accumulation of p62 and compromises association of LC3 with damaged lysosomes.Figure 5A HeLa cells stably expressing p97-GFP were control or UBE2QL1-depleted for 48 h and expression of p97-GFP was induced for the last 24 h. Cells were LLOMe or control treated for 1 h, fixed after a chase of 2 h, and stained with antibodies to K48 ubiquitin chains and LAMP1 as indicated. Note the reduction of the K48 and p97-GFP signals on damaged lysosomes in UBE2QL1-depleted cells. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 10 µm for inlays. B Automated quantification Shown are fold increases of Pearson correlation coefficients (P.C.C.) of p97-GFP and LAMP1 signals, normalized to untreated siCtrl. Data are from four independent experiments with ≥ 30 cells per condition. Data information: data are presented as mean ± SD. *P<0.05; ***P<0.001 (One-way-ANOVA with Bonferroni's multiple comparison test). C HeLa cells were control or UBE2QL1-depleted for 60 h, fixed after 1 h of LLOMe or control treatment and stained for LAMP1 and p62 and analyzed by confocal microscopy. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 5 µm for inlays. D Quantification Shown are fold increases of Pearson correlation coefficients (P.C.C.) of p62 on LAMP1 signals, normalized to untreated siCtrl. Graph represents data from three independent experiments with ≥ 30 cells per condition. Data information: data are presented as mean ± SD. *P<0.05; ***P<0.001 (One-way-ANOVA with Bonferroni's multiple comparison test). E HeLa cells were treated but fixed after additional 2 h of chase. Samples were stained for Galectin-3 (Gal3) to detect damaged lysosomes, and LC3B as marker for autophagosomes. Note the reduction of LC3B colocalization with Gal3 indicating impaired autophagosome formation. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 5 µm for inlays. F Quantification Shown are fold increases of Pearson correlation coefficients (P.C.C.) of LC3B and Gal3 signals, normalized to untreated siCtrl, representing LC3B colocalization with Gal3. Graph represents data from three independent experiments with ≥ 50 cells per condition. Data information: data are presented as mean ± SD. *P<0.05; ***P<0.001 (One-way-ANOVA with Bonferroni's multiple comparison test). G Western blot analysis of lysates of cells transfected with UBE2QL1 or control siRNA for 72 h, treated with 200 nM Bafilomycin A1 for 5 h or 250 µM LLOMe for 3 h as indicated and probed with an antibody specific for LC3A/B. GAPDH was probed as loading control.
Tumour-intrinsic PI3Kα alters tumour cell chemokine secretion and promotes the acquisition of tumour-associated inflammatory macrophage characteristics in the peritumoural tissue.Figure 7A-D Blood count of A, white blood cells, B, monocytes, C, granulocytes and D, lymphocytes in KPC mice, with cohort size in each group. E Basal level of IL-3 was quantified in KC;p110α+/+ (R211, A338 and A338L) and KC;p110α+/lox (A260 and A94L) cell lines isolated from mice primary tumours or lung metastases (L). Each point is a mean of n=3 independent values. F, IL-3 relative level (compared to vehicle) was determined in 3 KC (R211, A338 and A338L) cell lines isolated from mice primary tumours under treatment with vehicle (DMSO) or 1µM of BYL-719. n=3 independent values. G, Violin plot demonstrating the link between PI3Kα activation and activation of a selective population of γδLT in the PACA-AU cohort. h-jH-J H, Representative images and quantification of I, F4/80 positive cells and J, CD206 positive cells after the pharmacological inhibition of PI3Kα. Black arrowheads show positive immune cells. K,L Quantification of K, F4/80 and L, CD206 positive cells in KC and oncogenic PI3Kα mice. M,N Quantification of CD206 positive cells in M, low-grade and N, high-grade PanIN lesions after the genetic inactivation of PI3Kα in epithelial pancreatic cells. O, Quantification of CD206 positive in KPC mice at different stages of PDAC progression. P, treatment of macrophage cell line with tumour-cell conditioned medium. Q,R Quantification of secreted TNFα in indicated conditions. n=3 independent values for each cell line and treatment. S, Murine pancreatic tumour cells R211 were treated with vehicle, TNF-α 20 ng/mL, BYL-719 1 µM or both treatments and subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n= 4 independent values.
a) Quantification of mitochondria numbers from Atg18 control (Atg18KG03090/wild type) and mutant (Atg18KG03090/Df(3L)6112) or Atg7 control (Atg7d30/d14) and mutant (Atg7d77/d14) TEM images.figf5(a) Quantification of mitochondria numbers from Atg18 control (Atg18KG03090/wild type) and mutant (Atg18KG03090/Df(3L)6112) or Atg7 control (Atg7d30/d14) and mutant (Atg7d77/d14) TEM images. Mitochondria were quantified from 2 distinct 25 μm2 regions per cell from 2 cells per animal from at least 3 different animals per genotype. (b,c) Midguts dissected from either early third instar larvae (b) or at puparium formation (c) that express GFP-labelled mitochondria in all cells and contain an Atg1Δ3D loss-of-function mutant cell clone (lacking RFP). Wild-type control cells possess stronger RFP and heterozygous cells have weaker RFP. Representative images are shown. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 0 μm.