EMBO/sd-character-level-ner huggingface dataset:
Figure 2(A) Consecutive in vivo imaging of the same apical dendrites from layer V pyramidal neurons in the somatosensory cortex over 46 days reveals formation and elimination of dendritic spines (white and empty arrowheads, respectively) in WT and APP-KO mice. Prior to the exposure to environmental enrichment (EE), all mice were housed in standard conditions. Scale bar - 10 μm. (B-C) Graphical representations of the relative spine density and spine elimination over the period of the exposure of mice to EE. Non-linear regression (F test) has been used for fitting the data points. Repeated one-way ANOVA was performed followed by Dunnett test. WT n=5 mice and APP-KO n=6 mice; * p<0.05, ** p<0.01, NS - no significant difference.
Unless otherwise stated all experiments were repeated three times and data shown are representative.figf4(a) Beclin-1 alone (lanes 1-4) or Beclin-1 and ATG14L (lanes 5-8) were overexpressed in HEK293 cells. Beclin-1 was purified either by direct immunoprecipitation (lanes 1-4) or by ATG14L immunoprecipitation (lanes 5-8). Immunoprecipitated samples were subjected to an in vitro ULK1 kinase assay with increasing amounts of ULK1. Reactions were immunoblotted with the indicated antibodies. Black line denotes discontinuous lanes from the same gel. Two unique experiments were performed. (b) Beclin-1 alone or bound to ATG14L was purified as described in a. Equal amounts of ULK1 were added to each complex and reactions were quenched at the indicated time points. Western blotting was performed with the indicated antibodies. (c) An ATG14L-FLAG-6His-inducible U2OS cell line was induced for 16 h in the presence of amino acids. Endogenous Beclin-1 was immunoprecipitated and immunoblotted as in Fig. 3a. ATG14L input levels were detected by immunoblotting. Two unique experiments were performed. (d) HEK293 cells transfected with either ATG14L or Beclin-1, or both, in conjunction with ULK1, were immunoprecipitated as indicated and blotted with the indicated antibodies. (e) HEK293 cells were transfected with Beclin-1 and ULK1 in the presence of ATG14L WT or ATG14LΔCCD, which is defective in Beclin-1 binding, under nutrient-rich conditions. Lysates were resolved by SDS-PAGE and blotted with the indicated antibodies. (f) HEK293 cells were transfected with ULK1 and Beclin-1 in conjunction with either ATG14L WT, or one of two mutants (ΔBATS,ΔN) that are defective in phagophore localization. Samples were handled as in e. Uncropped images of blots are shown in Supplementary Fig. S4. Lysates were resolved by SDS-PAGE and blotted with the indicated antibodies.
Low NAV3 abundance correlates with shorter survival of breast cancer patients.Figure 7A Representative immunohistochemistry images of NAV3 in a normal breast tissue, as well as in the indicated molecular subtypes of breast cancer. B Tissue microarrays were used to stratify 288 breast cancer patients for overall survival according to NAV3 abundance. C Immunohistochemistry was used to score NAV3 expression and stratify 323 breast cancer patients according to the indicated subtypes and tumor parameters. D, E Kaplan-Meier curves depicting survival of 1,471 estrogen receptor-positive breast cancer patients (D) and 1,405 lung cancer patients (E) stratified according to NAV3 abundance.
Mathematical model supports a role for Dll4 in controlling cell recruitment into IAHCs.Figure 7Schematics of the probabilistic model of IAHC formation. The processes considered in the model are (1) initiation of cluster, (2) cell division event, (3) recruitment event, and (4) cluster detachment. Each process in the model is described as a typical time scale (Tcluster lifetime, Tcell division, Trecruitment). In case of αDll4 treatment, the final five hours of the simulation are assumed to have a different recruitment time. See details of the simulation in the methods. Heat map showing the probability (or p-value) that the simulation produces mean cluster size and fraction of colored cells that match those observed experimentally for the IgG treatment. This probability is calculated for different values of cluster lifetimes and mean recruitment times (values in x and y). 4000 simulations were performed for each set of parameters (each point in the heat map). The most likely parameter values (yellow) correspond to a cluster lifetime of 21 hours and mean recruitment time of 11 hours. Red dashed line indicates the cross-section with the most likely cluster lifetime (21 hours). (C) A graph showing the probability that the simulation produces mean cluster size and fraction of colored cells that match those observed experimentally for the αDll4 treated clusters (blue line). Here, the cluster life time is taken to be 21 hours (taken from b) and the probability to match the experimental data is calculated for different recruitment times in the final 5 hours of the simulation (x-axis). The most likely mean recruitment time for the αDll4 treatment is 5 hours (peak of blue line). For comparison, the most likely mean recruitment time for the IgG treatment is 11 hours (Red dashed line, corresponding to the red dashed in (B)). Hotelling's T-Squared test. Examples of simulations of IAHC distributions for the IgG (left) or αDll4 (right) treated samples. Simulations were performed with the most likely parameter values described in b and c. Note that only clusters that have at least one colored cell are shown (i.e clusters with non-colored cells are not represented). Snapshots of Movie EV5. Time Lapse of embryonic organotypic slice from a CD41:YFP: H2B-GFP reporter mouse showing the recruitment of CD41:YFP negative cells (magenta central point-tracking analyses) migrating towards the IAHC (CD41:YFP+ cells). Green central point and relative tracking identify cells with non-directional movement. Scale bar: 5μm. Time expressed in hh:mm:ss.
Figure 1A-B Surface representations of the macrodomain structures for the human macroH2A isoforms macroH2A1.1 (PDB ID 1ZR3) and macroH2A2 (PDB ID 6FY5), respectively, with electrostatic potential included. A yellow-framed box indicates the location of the major surface pocket accommodating ADP ribose in macroH2A1.1. C-D Close-up view of the nucleotide binding region of ADP ribose bound by human macroH2A1.1 (C) overlayed with the corresponding region of macroH2A2 in orange (D). An important structural difference in macroH2A2 that distorts de nucleotide binding loop is highlighted in magenta, glycines in the nucleotide binding loop are depicted as spheres (G312 and G314). The ADP-ribose ligand is included in D (shaded light gray) to facilitate orientation and comparison. Dashed lines indicate polar interactions.
Crystal structure of the Atg12–Atg5 conjugate bound to Atg16N.Figure 1(A) Ribbon representation of the Atg12-Atg5 conjugate bound to Atg16N. Atg12, Atg5 and Atg6 are coloured salmon pink, green and orange, respectively. Atg12 Phe 185 and Gly 186 and Atg5 Lys 149 are shown with a stick model. The disordered C‐terminal region of Atg12 (residues 182-184) is indicated with a broken line. The left figure was obtained by a 90° rotation of the right figure along the vertical axis. The amino‐ and carboxy‐termini are denoted as N and C, respectively. All of the figures representing the molecular structures were generated with PyMOL [ 26]. (B) Stereo view of the detailed interactions between Atg12 and Atg5. The side chains of the residues involved in the Atg12-Atg5 interaction are shown with a stick model, in which nitrogen and oxygen atoms are coloured red and blue, respectively. (C) Electron density map of the isopeptide linkage between Atg12 Gly 186 and Atg5 Lys 149. The simulated annealing Fo-Fc difference Fourier map was calculated by omitting Atg12 Phe 185 and Gly 186 and Atg5 Lys 149, and is shown with black meshes at 5.0σ. (D) Superimposition of the Atg5‐Atg16N complex structure (PDB ID 2DYM) on that of the Atg12-Atg5‐Atg16N complex. The Atg12-Atg5‐Atg16N complex is coloured red, while the Atg5‐Atg16N complex is coloured grey. Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model. (E) In vivo analyses of the Atg12 and Atg5 mutants for studying the functional significance of the non‐covalent Atg12‐Atg5 interactions. Yeast cells with or without starvation were lysed and subjected to urea SDS-polyacrylamide gel electrophoresis (PAGE) followed by western blotting. Ape1, aminopeptidase I; mApe1, mature form of Ape1; PE, phosphatidylethanolamine; prApe1, preform of Ape1; WT, wild‐type.
Figure 6(A) The effect of over-expression of the indicated host proteins on EBOV RNA synthesis was assessed using an EBOV MG assay. HEK293T cells were transfected with plasmids expressing components of the MG assay and either 12.5 or 125 ng of expression plasmids for the indicated host factors. Empty expression plasmid (vector) served as a control. Luciferase activity was measured 48h post-transfection and fold MG activity was calculated relative to a no VP30 control. Data information: , the data represent the mean ± S.D. from one representative experiment in which each transfection condition was performed in triplicate (n=3). The experiment was reproduced in two additional, independent experiments Statistical significance was calculated using ANOVA with Tukey's multiple comparisons test. ****p<0.00005; ***p<0.0005, **p<0.005, *p<0.05. (B) MG activity upon transient expression of GFP or GFP-fused PPxPxY-containing peptides. Plasmids expressing GFP fused peptides were transfected at 12.5 and 125 ng amounts. Statistical significance was calculated relative to the GFP control for each concentration. Data information: , the data represent the mean ± S.D. from one representative experiment in which each transfection condition was performed in triplicate (n=3). The experiment was reproduced in two additional, independent experiments Statistical significance was calculated using ANOVA with Tukey's multiple comparisons test. ****p<0.00005; ***p<0.0005, **p<0.005, *p<0.05. (C) MG activity upon knockdown of host genes. HEK293T cells were transfected with scrambled siRNA or siRNA targeting RBBP6, hnRNP L, hnRNPUL1 or PEG10. Twenty-four hours post-transfection, cells were transfected with MG assay plasmids including VP30 at 12.5 and 25ng doses. Data information: , the data represent the mean ± S.D. from one representative experiment in which each transfection condition was performed in triplicate (n=3). The experiment was reproduced in two additional, independent experiments Statistical significance was calculated using ANOVA with Tukey's multiple comparisons test. ****p<0.00005; ***p<0.0005, **p<0.005, *p<0.05. (D) MG activity was assessed as in panel C upon single or double knockdown of RBBP6 and hnRNP L. Data information: the data represent the mean ± S.D. from one representative experiment in which each transfection condition was performed in triplicate (n=3). The experiment was reproduced in two additional, independent experiments Statistical significance was calculated using ANOVA with Tukey's multiple comparisons test. ****p<0.00005; ***p<0.0005, **p<0.005, *p<0.05.
USP42 antagonizes R-spondin/LGR and promotes FZD and LRP6 protein turnover A, Cell surface biotinylation assay performed in HEK293T cells transfected as indicated and treated with 10 μM MG132 to prevent proteasomal degradation.Figure 3A, Cell surface biotinylation assay performed in HEK293T cells transfected as indicated and treated with 10 μM MG132 to prevent proteasomal degradation. Consecutive immunoprecipitations were carried out as indicated in the scheme. The Avidin pulldown was carried under denaturing conditions (1st IP). The unrelated receptor CD147 was used as loading control for the avidin pull down, and CUL1 for the total input of cell lysate. Co-immunoprecipitation experiments in HEK293T cells transfected with the indicated constructs and treated for 6 h with Bafilomycin A1. Cells were co-treated with RSPO1 (B) conditioned medium. In (B), co-immunoprecipitated cytoplasmic USP42 (USP42∆NLS) protein levels were quantified relative to immunoprecipitated ZNRF3. ZNRF3 levels in the input were quantified relative to tubulin. C, Co-immunoprecipitation experiments in HEK293T cells transfected with the indicated constructs and treated for 6 h with Bafilomycin A1. Cells were co-treated with RSPO3 (C) conditioned medium. In (C), co-precipitated LGR4 protein levels were quantified relative to immunoprecipitated ZNRF3∆Ring. Representative blots of n = 3 independent experiments are shown. D, Scheme showing the proposed function for USP42 towards ZNRF3. E, Immunofluorescence microscopy of HEK293T cells transfected with SNAP-FZD5 and the indicated constructs. Cells were incubated with SNAP-surface-549 for 15 min and chased for another 10 minutes prior fixation. Representative images from one out of two independent experiments are shown. The white arrows show SNAP-FZD5 undergoing plasma membrane clearance. Scale bar = 20 μm. F,G, Western blots of lysates from HEK293T cells transfected with the indicated constructs. The asterisk marks an unspecific band. Representative blots from at least three independent experiments are shown. H, Western blots of lysates from HEK293T cells transfected with the indicated siRNAs. Where indicated, cells were treated for 6 h with RSPO3 conditioned medium. Representative blots from n = 4 independent experiments are shown.
Figure 1A. qPCR analysis of Ahr expression in B cell subsets purified from C57Bl/6 mice. Ahr expression was normalized to Hprt1. n = 3-5 independent experiments; mean ± SD. B. qPCR analysis of Ahr expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 4h as indicated. Ahr expression was normalized to Hprt1. Ahr expression among groups was normalized to Medium. n = 4 independent experiments; mean ± SEM; one-way ANOVA, Dunnett's multiple comparison test. C. qPCR analysis of Ahr expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 4h with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Ahr expression was normalized to Hprt1. Ahr expression among groups was normalized to Medium. n = 3 independent experiments; mean ± SEM; one-way ANOVA, Tukey's multiple comparison test. D. Western blot of protein extracts from splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 4h with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Values above the blots indicate AhR protein quantification obtained by densitometry, normalized to β-actin and compared to Medium sample. Sample A and B indicate 2 independent replicates. E. Quantification by densitometric analysis of the results shown in C. n = 2 independent experiments; mean ± range. F. qPCR analysis of Ahr expression in purified splenic B cell subsets isolated from C57Bl/6 mice and cultured as indicated for 4h. Ahr expression was normalized to Hprt1. n = 2 independent experiments; mean ± range. G. qPCR analysis of Ahr expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for the indicated time points with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Ahr expression was normalized to Hprt1. Ahr expression among groups was normalized to Medium. n = 5 independent experiments; mean ± SEM; one-way ANOVA, Dunnett's multiple comparison test.
Figure 3A. Higher induction of inflammatory cytokine mRNA levels in LPS-stimulated VI10 PDFs than in CFs. The expression level in resting conditions of the PDFs was set as 1. Differences between knockdown and control with p values ≤ 0.05 (Student's t test) are indicated with an asterisk. B. as in A. for patient VII1. C. knockdown of PPP1R13L in CFs resulted in increased mRNA expression levels of inflammatory mediators. Differences between knockdown and control with p values (Student's t test) are indicated with asterisks in the graph.
Ubqln1 punctate distribution is dependent on Ubqln4.Figure 3(A) Confocal images of Hela cells transfected with GFP‐S‐Ubqln1 and also transfected with either control or Ubqln4 siRNA. The cells were either untreated or were transferred to starvation medium for 1 h. (B) Quantification of the number of GFP‐S‐Ubqln1 WT puncta per cell in (A). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. *P‐value=0.011, **P‐value=0.0005 and ***P‐value=0.0001. (C) Depletion of Ubqln4 with siRNA in (A) was verified by western blot using anti‐Ubqln1, anti‐Ubqln4 and anti‐tubulin antibodies. (D) Hela cells transfected with GFP‐S‐Ubqln1 and also transfected with either control or Ubqln4 siRNA were starved for 1 h. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐LC3 antibodies. (E) Confocal images of GFP‐S‐Ubqln1 WT in Hela cells transfected with RFP, RFP‐Ubqln4 or RFP‐Ubqln4 ΔUBL. (F) Quantification of the number of GFP‐S‐Ubqln1 WT puncta per cell in (E). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. *P‐value=0.0001 and **P‐value=0.0003. GFP, green fluorescent protein; IP, immunoprecipitation; LC3, microtubule‐associated protein light chain 3; UBL, ubiquitin‐like; Ubqln, Ubiquilin; WCL, whole‐cell lysate; WT, wild‐type.
a) Formation of endogenous ATG12 vesicles in CALM knockdown HeLa cells in basal (BC) and serum.f3(a) Formation of endogenous ATG12 vesicles in CALM knockdown HeLa cells in basal (BC) and serum starvation conditions. Confocal pictures are shown with arrows indicating ATG12 vesicles in starvation conditions. Data are from one representative experiment, out of three independent experiments. Data shown as mean ±s.e.m. (n≥500 cells; *P0.01; two-tailed t-test). Scale bars, 5 μm. (b) Co-localization between GFP-ATG16L1 and internalized cholera toxin subunit B conjugated with Alexa555 (CTX; 20 min) in CALM knockdown HeLa cells. Confocal pictures are presented with magnified areas showing the co-localization between ATG16L1 and CTX in greater detail. Scale bars, 5 μm. The Pearson's coefficient between ATG16L1 and cholera toxin is shown. Data are representative of three independent experiments and shown as mean ±s.d. (n≥20 cells; *P0.05; two-tailed t-test). (c) Size of endogenous ATG12 vesicles in CALM knockdown HeLa cells in starvation conditions. Confocal pictures are shown with arrows indicating ATG12 vesicles and # indicating CALM knockdown cells. Data are representative of three independent experiments and shown as mean ±s.d. (n≥100 vesicles; *P0.05; two-tailed t-test). (AU, arbitrary unit). Scale bars, 5 μm. (d) Live-cell imaging of ATG16L1-GFP in CALM knockdown HeLa cells. ATG16L1 pictures from various time points of a 5-min movie are shown in inverted grey style. Arrows indicate ATG16L1 vesicles. The number of fusion events per 5 min is shown. Data are representative of five movies and shown as mean ±s.d. (*P0.05; two-tailed t-test). Scale bars, 5 μm. (e) In vitro fusion assay of post-nuclear supernatant from HeLa cells expressing either GFP-ATG16L1 or mStrawberry-ATG16L1 in control and CALM knockdown conditions. Confocal pictures are shown where ATG16L1-mStrawberry signal is shown in purple to enable better visualization. Fused vesicles appear in white. The ATP-negative condition, which prevents SNARE-dependent fusion, is also shown as a control for the reaction. Magnified areas are shown to allow visualization of the vesicles. The percentage of fused vesicles is represented. n=numbers of vesicles scored per field (a minimum of five fields were analysed per condition). Data are representative of two independent experiments and shown as mean ±s.d. (n≥100 vesicles). Scale bars, 5 μm. (*P0.05; two-tailed t-test).
Transfer of HFD-fed NOD2−/− microbiota contributes to inflammation and insulin resistance.Figure 7A Hierarchical cluster tree built using UPGMA (unweighted pair group method with arithmetic mean) based on the unweighted UniFrac distance matrix. Each branch represents a numbered mouse from each group of WT donor, WT-R (WT recipient, which are WT germ-free mice that received WT donor flora), NOD2−/− donor, or NOD2-R (NOD2 recipient, which are WT germ-free mice that received NOD2−/− donor flora). Branches in the UPGMA tree are colored according to their jackknife support: red, 75-100%; yellow, 50-75%; green, 25-50%; and blue, < 25% support.B Calculation of the unweighted UniFrac matrix similarity index showed the level of successful transfer of the microbiome to recipient mice evidenced by results comparing the similarity within each group to the union (U) between different conditions. Conditions with different letters (a, b c, d, e) in the similarity index denote a statistical difference compared to all other conditions without the same letter, where the minimal statistical values for all comparisons are: aP = 0.01, bP = 0.04, cP = 0.004, dP = 0.0001, and eP = 0.002. C Relative abundance of phyla in the cecum from each recipient mouse. D Body mass in weight-matched HFD-fed WT-R (n = 5) and NOD2-R (n = 4) mice, 5 weeks after colonization of germ-free mice. E, F Fasting bloodglucose (E) and HOMA insulin resistance (IR) index (F) in weight-matched HFD-fed WT-R (n = 5) and NOD2-R (n = 4) mice, 5 weeks after colonization of germ-free mice, *P = 0.02 and **P = 0.0001. G Blood glucose and quantification of the AUC during a PTT in HFD-fed WT-R (n = 7) and NOD2-R (n = 6) mice, *P = 0.01. H, I Quantification of immune cell and inflammatory markers (H) and PRRs (I) in adipose tissue of weight-matched HFD-fed WT-R (n = 5) and NOD2-R (n = 4) mice, *P = 0.01, **P = 0.001, and ***P = 0.0001. J Quantification of metabolic transcripts in the liver of weight-matched HFD-fed WT-R (n = 5) and NOD2-R (n = 4) mice, *P = 0.04.
Figure 1All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.A) Immunohistochemistry analysis of small intestinal sections, using the indicated antibodies. All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.B) Western blot analysis with protein extracts obtained from ex vivo-purified intestinal crypts and probed with the indicated antibodies. All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.C) Haematoxylin and eosin staining of sections prepared from different intestinal tracts. All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.D) Immunohistochemistry analysis of small intestinal sections, using specific antibodies for the proliferation marker KI67. All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.E) Expression analysis by qRT-PCR of the proliferation related markers indicated in the figure using RNA extracted from intestinal crypts. Graphs show mean ± SD for three replicates.
Low LC3-II expression in MoDCs infected with P.Fig 7A) Western blot detection of LC3 in MoDCs infected with Pg381 and mutant strains for 12 hours. B) The mean ±standard deviation of LC3-II intensity represents three different experiments and the values were analyzed using Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons. C) Blot shows the autophagy flux test after blocking the lysosomal fusion with Bafilomycin in MoDCs. Cells were treated with Bafilomycin for 4 hours at 4nM. D) LC3-II intensity in MoDCs infected with Pg381 strains with and without Bafilomycin. The analysis of the intensity of three different experiments used Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons.