EMBO/sd-character-level-ner huggingface dataset:
BI‐1 regulates Beclin‐1‐dependent autophagy by controlling JNK activation.Figure 4(A) BI‐1 WT and KO MEFs were treated with EBSS for 2 h, and the levels of phospho‐p70S6k were determined by western blot analysis. Total p70S6k is also shown. (B) LC3 was monitored by immunofluorescence in cells treated with EBSS for 3 h in the presence or absence of 10 μM JNK inhibitor SP600125. Mean and standard deviation are presented (N=3). Student's t‐test was used to analyse statistical significance, *P0.001 and **P0.0001. (C) BI‐1 WT and KO cells were treated with EBSS for indicated time points, in the presence or absence of 10 μM of the JNK inhibitor SP600125. Levels of phosphorylation of JNK (pJNK) and LC3‐II were determined by western blot. The levels of total JNK and Hsp90 are shown as control (N=4). Image was assembled from cropped lanes of the same western blot analysis of the same gel. (D) BI‐1 WT and KO cells were treated with EBSS for indicated time points in the presence or absence of 10 μM SP600125, and the electrophoretic shift associated with BCL‐2 phosphorylation was monitored by western blot. (E) BI‐1 WT and KO cells were co‐transfected with expression vectors for Beclin‐1-MYC, BI‐1-HA, and BCL‐XL-FLAG. After 24 h, cells were treated with EBSS for 2 h or left untreated. The association of MYC‐tagged expressed Beclin‐1 and BCL‐XL-FLAG was assessed by immunoprecipitation of Beclin‐1 followed by western blot analysis. (F) 293T cells were co‐transfected with expression vectors for Beclin‐1-MYC, BI‐1-HA, and BCL‐XL-FLAG. Cell extracts were prepared in CHAPS buffer and Beclin‐1-MYC immunoprecipitated, and the possible co‐precipitation of BI‐1-HA, and BCL‐XL-FLAG was assessed by western blot analysis (N=3). Asterisks indicate BI‐1 oligomers. (G) (G) 293T cells were co‐transfected as described in (F) and BI‐1-HA was immunoprecipitated, and the possible co‐precipitation of Beclin‐1-MYC, and BCL‐XL-FLAG determined by western blot analysis (N=3). Figure source data can be found with the Supplementary Information.
a) hlh-30 quantitative rtPCR showing increased expression of the C.figf8(a) hlh-30 quantitative rtPCR showing increased expression of the C. elegans TFEB gene hlh-30 over a time course of starvation in wild-type animals followed by a rapid decrease to the basal level following re-feeding of the animals (mean±s.e.m. of n = 3). *P≤0.05 (t-test, compared with wild-type at t = 0 h). (b) hlh-30 3' UTR quantitative rtPCR showing expression of hlh-30 after 12 h starvation in wild-type and hlh-30(tm1978) animals (mean±s.e.m. of n = 3). *P≤0.05 (t-test, compared with wild-type starved). (c) Quantification of oil red O stain in starved animals relative to well-fed counterparts (mean±s.e.m. of n = 3). **P≤0.01 (t-test, compared with wild-type starved). (d-g) Representative micrographs of wild-type and hlh-30 animals after 8 h starvation and stained with oil red O. (h-k) Representative TEMs of wild-type and hlh-30 animals after 24 h starvation. IEC, intestinal epithelial cell; EPI, epidermis; BB, brush border; GON, gonad; BWM, body wall muscles, aj, apical junction; ld, lipid droplet. Scale bar, 2 μm. (l) Quantitative rtPCR of starvation-induced genes in wild-type and hlh-30 animals after 12 h starvation (means ± s.e.m of n = 3). *P≤0.05; **P≤0.01; ***P≤0.001 (t-test, compared with wild-type starved). (m,n) hlh-30 is required for starvation-induced lifespan extension. One representative experiment of 2 independent trials; error bars represent mean±s.e.m. Median survival (MS) (wild-type fed) = 10 d; MS (wild-type starved) = 17 d, P0.0001 versus fed using the Log-rank test; MS (hlh-30 fed) = 7 d; MS (hlh-30 starved) = 9 d, P0.0001 versus fed. (o) L1 arrest assay showing survival of wild-type and hlh-30 starved animals relative to non-starved conditions (mean±s.e.m. of n = 3). ***P≤0.001 (t-test, compared with wild-type starved).
Figure 7A In vivo bioluminescent imaging analysis of intracranial tumor growth in mice bearing GBM xenografts derived from the T387 GSCs expressing shUSP33 or shNT. Two thousand GSCs transduced with luciferase and shUSP33 or shNT were implanted into mouse brains through intracranial injection. Representative bioluminescent images on day 7, day 15 and day 30 post-transplantation of GSCs were shown. Disruption of USP33 delayed tumor growth of intracranial GBM xenografts. B Kaplan-Meier survival curves of mice bearing GBMs derived from T387 GSCs expressing shUSP33 or shNT from (A). Disruption of USP33 extended the survival of mice bearing GSC-derived GBM tumors. (n= 5 mice for each group; shUSP33#42 vs shNT, p = 0.0185; shUSP33#45 vs shNT, p = 0.0027; two-tailed log-rank test). C, Immunofluorescent analysis of the apoptotic marker cleaved caspase 3 in xenografts derived from T387 GSCs expressing shUSP33 or shNT. Disruption of USP33 in GSCs dramatically elevated apoptosis in GBM tumors. Scale bar, 80 μm. statistical quantifications (D) of the apoptotic marker cleaved caspase 3 in xenografts derived from T387 GSCs expressing shUSP33 or shNT. (n = 5 tumors for each group; ***, p < 0.001; mean ± s.e.m.; two tailed unpaired t-test). E, F Immunofluorescent analysis (E) and statistical quantifications (F) of the cell proliferation marker Ki67 in xenografts derived from T387 GSCs expressing shUSP33 or shNT. Disruption of USP33 in GSCs reduced cell proliferation in GBM xenografts. Scale bar, 80 μm. (n = 5 tumors for each group; ***, p < 0.001; mean ± s.e.m.; two tailed unpaired t-test). G, H Immunofluorescent analysis (G) and statistical quantifications (H) of the vascular endothelial cell marker Glut1 in xenografts derived from T387 GSCs expressing shUSP33 or shNT. Disruption of USP33 in GSCs reduced vessel intensity in GBM xenografts. Scale bar, 80 μm. (n = 5 tumors for each group; ***, p < 0.001; mean ± s.e.m.; two tailed unpaired t-test).
Figure 3Male, GADD45β+/+ (WT; n=6) or GADD45β-/- mice (KO; n=4) were fed ad libitum (fed) or fasted for 24h (fasted), and subsequently refed for 24h. O2 consumption rate (A), CO2 production rate (B) and respiratory exchange ratio (C) were measured by indirect calorimetry. In a distinct cohort, male GADD45β+/+ (WT; n=5) or GADD45β-/- mice (KO; n= 8) were fed ad libitum (fed) or fasted for 24h (fasted). Serumnon-esterified fatty acids (D) and ketone bodies (E) as well as serum (F) and liver (G) triglycerides (TG) were measured. Serum TG (H), NEFA (I), and blood glucose (BG, J) concentrations during an oral lipid tolerance test in overnight fasted GADD45β+/+ (WT; n = 5) or GADD45β-/- (KO; n=5) mice. Bloodglucose excursion during and intraperitoneal insulin tolerance test (K) as well as fasting bloodglucose (L), seruminsulin (M), and HOMA-IR (N) in GADD45β+/+ (WT; n = 6) or GADD45β-/- mice (KO, n = 9) chronically fed a normal- (NFD) or high (HFD) fat diet. Data are mean ± SEM.
Akt activation in response to Salmonella requires FAK.Figure 3(A) WT and FAK−/− PEMs were incubated for 0-5 hours with ΔinvG Salmonella before immunoblotting with the indicated antibodies. (B) WT PEMs were pretreated for 1 hour with the FAK-kinase inhibitor PF-228 (0.5 µm) before incubation with ΔinvG Salmonella for 5 hours. Lysates were immunoblotted with the indicated antibodies. (C-D) WT or FAK−/− macrophages were incubated with either the ΔΣΠΙ1ΔΣΠΙ2 strain (C) or LPS-coated beads (D) for 0-5 hours before immunoblotting with the indicated antibodies. In panels C and D, WT PEMs were also incubated with ΔinvG (5 h) for comparison of Akt activation. Vertical white lines in panel D indicate non-contiguous lanes generated from a single exposure. E) Levels of phosphorylated proteins were quantified by densitometry, normalized to the amount of total protein present in each sample, and expressed relative to the basal level in uninfected cells. Values are means ± SEM, N = 3, *p<0.05. Dashed line drawn at 1 indicates basal levels of activation. (F) WT PEMs expressing HA-Akt were either left untreated or incubated with ΔinvG Salmonella for 5 hours. Lysates were subjected to immunoprecipitation (IP) with anti-HA antibodies before immunoblotting for FAK and HA. In parallel, lysates were immunoblotted for FAK, HA and ERK1/2 to control for the level of expression. G) WT and FAK−/− macrophages expressing HA-Akt were stained with anti-HA and anti-LAMP1 before analysis by confocal microscopy. White boxes show enlarged regions in lower panel. Arrows indicate HA+LAMP+ Salmonella evident in WT PEMs. (H) The percentage of ΔΣΠΙ1ΔΣΠΙ2 (first grouping) or ΔinvG (second grouping) Salmonella co-localizing with HA and LAMP-1 5 h post-infection. At least 100 bacteria were counted per condition. Values are means ± SEM, N = 3, *p<0.05.
High‐resolution identification and classification of agonist‐liganded ARBEs in vivoAR ChIP‐exo defines four types of high‐confidence agonist‐liganded ARBEs.Figure 2A ARChIP‐exo defines four types of high‐confidence agonist‐liganded ARBEs. Numbers indicate motif counts. The first two columns of each ARBE panel show normalized tags distribution over ARBEs on the forward (blue) and reverse (red) strands, separately. Forward strand refers to the motif‐containing strand. The aggregated tag density is shown at the top of the raw signal plots. The third column of each ARBE panel represents the bound motif sequences ordered as in the left two columns. B Comparison of tag density shows the four types of ARBEs are located between one common upstream border on the forward strand and one common downstream border on the reverse strand. Other border signals on ARBEs are also shown. The protected conserved nucleotides of each ARBE have the same distance to both common borders. C Clustering of ARBE1 based on the motif probability matrix of the extended second‐half ARBE.
Schematic presentation of the disease associated / homeostatic phenotypes observed in Grn-/- mice versus Trem2-/- and the potential consequences for a therapeutic window of microglial modulation.Figure 6Schematic presentation of the disease associated / homeostatic phenotypes observed in Grn-/- mice versus Trem2-/- and the potential consequences for a therapeutic window of microglial modulation.
Increased AMPK/AAK2 and PKA/KIN-1/2 activity occur in the long-lived age-1; gas-1 mutants.Figure 3Schematic representation with overlaid Molecule Activity Predictor (MAP) analysis of (A) AAK-2/AMPK- in age-1; gas-1 versus gas-1 mutants. Color intensity corresponds to effect intensity or prediction strength. Schematic representation with overlaid Molecule Activity Predictor (MAP) analysis of (B) KIN-1/PKA-regulated metabolic processes in age-1; gas-1 versus gas-1 mutants. Color intensity corresponds to effect intensity or prediction strength. (C) Immunoblot analysis of phospho-AAK-2 in wt, age-1, gas-1 and age-1; gas-1 mutant nematodes (actin loading control). Numbers correspond to band densitometries across 3 biological replicates. (D) Representative lifespan of age-1; gas-1 double mutant and age-1; aak-2; gas-1 triple mutant nematodes with average median lifespans ± SEM from all replicates indicated beneath representative curves. (E) Lifespan assay of gas-1 mutant animals compared to gas-1; AAK-2 overexpressing (O/E) nematodes and their wt counterparts. Inset: immunoblot analysis of phospho-AAK-2 in gas-1 (g) and gas-1; AAK-2 O/E (g; a O/E) animals (actin loading control), showing the overexpressed protein (lower band) and the endogenous one (upper band). (F) Left panel: Immunoblot analysis of phospho-KIN-1 substrates in young adult nematodes. Numbers correspond to band densitometries across 2 biological replicates. Right panel: immunoblot analysis of phospho-KIN-1 substrates in age-1; gas-1 animals grown on bacteria carrying an empty vector (EV) or expressing kin-1 RNAi. Two different kin-1 RNAi clones (g1, b5) were used in this experiment. Numbers correspond to band densitometries of the presented immunoblot. Dashed rectangles: band corresponding to downregulated KIN-1 phospho-substrate. (G) Representative lifespan assay of wt and age-1; gas-1 nematodes fed with empty vector (EV) and kin-1 RNAi bacteria with average median lifespans ± SEM from all replicates indicated beneath representative curves. ATP measurements in: (H) age-1(hx546); gas-1(fc21) (a; g) and age-1(hx546); aak-2(ok524); gas-1(fc21) (a; aa; g) mutant nematodes; ATP measurements in: (I) age-1; gas-1 (EV) (a; g (EV)) and age-1; gas-1; kin-1 (RNAi) (a; g; kin-1(RNAi)). Error bars: Mean ± SEM, n=4, *p value <0.05, Mann Whitney U test. (J) Left panel: curves represent FRAP in age-1; gas-1 animals grown on RNAi from larval L1 (or from egg for fzo-1 RNAi) to L4 stage. Each point: mean recovery of bleached regions (2-5 regions per animal, 10-20 animals per condition) ± SEM. Right panel: Maximum (total) recovery per condition are shown, ± SEM. *p value< 0.05, **p value< 0.01, ****p value< 0.0001, Kruskal-Wallis test, Dunn's post-hoc correction.
Figure 2Figure 2. SLFN11 forms a complex with RPA. (A) HEK293 cells stably expressing SFB-tagged (S-tag, Flag epitope tag, and streptavidin-binding peptide tag)-SLFN11 were used for TAP of protein complexes. Tables are summaries of proteins identified by Mass spectrometry analysis. Letters in bold indicate the bait proteins. . (B) SLFN11 interacts with RPA, but not with the Morc3 control protein. HEK293 cells were transiently transfected with plasmids encoding SFB-tagged SLFN11 together with plasmids encoding Myc-tagged RPA or Morc3. Cell lysates were immunoprecipitated with S beads and Western blot analysis was performed with anti-Flag and anti-Myc antibodies. (C-D) Association of endogenous SLFN11 with RPA in SF268 (C) or DU145 (D) cells was performed by co-immunoprecipitation using anti-SLFN11 or anti-RPA2 antibody. Nuclease-treated cell lysates were incubated with protein A agarose beads conjugated with indicated antibodies and Western blot analysis was performed according to standard procedures. (E) Direct binding between recombinant GST-tagged-SLFN11 and MBP-tagged-RPA1. Upper panel: SLFN11 was detected by immunoblotting. Lower panel: Purified proteins visualized by Coomassie staining. (F) Schematic representation of wild-type and deletion mutants of SLFN11 used in this study. (G) HEK293 cells were transfected with indicated plasmids. Cell lysates were immunoprecipitated with S beads and Western blot analysis was performed with anti-Flag and anti-Myc antibodies.
CA VII expression during human brain development.Figure 6CA VII expression during human brain development. Line plots show the log2-transformed exon array signal intensity in the neocortex (NCX) and hippocampal formation (HIP) from the early fetal period to late adulthood. The solid line between periods 7 and 8 separates the prenatal from the postnatal period. CA VII expression during human brain development. Line plots show the log. Line plots show the normalized ratio of absolute copy number of CA VII to Gapdh by droplet digital PCR (ddPCR, right) in the neocortex (NCX) and hippocampus (HIP) from the early fetal period to late adulthood. The solid line between periods 7 and 8 separates the prenatal from the postnatal period.
Figure 4Quantification of total length of vessels (A) in vehicle and glufosinate (20mg/kg) treated mice (n=6). Quantification of CD31+ tumor vessel area (B) and of αSMA+ pericyte-covered vessels over the total number of CD31+ vessels (with representative images) (C) in vehicle and glufosinate (20mg/kg) treated mice (n=6). (D) Quantification and representative images of Lectin+ tumor vessel area over the total number of CD31+ vessels in vehicle and glufosinate (20mg/kg) treated mice (n=6). (E) Quantification and representative images of PIMO+ tumor hypoxic areas in vehicle and glufosinate (20mg/kg) treated mice (n=6). FACS quantification of TCRbeta+ CD8+ cytotoxic T cells in tumors of vehicle and glufosinate-treated mice (F). Evaluation of CD69 (G) and CD25 (H) proteins, expressed as Mean Fluorescence Intensity (MFI), was determined by flow cytometry on the surface of responders CD8+ T cells (n=4). (I) Quantification of secreted IFNγ in interstitial tumoral fluid of vehicle and glufosinate-treated mice (n=6).
Figure 3A) Illustration of the experimental procedure. Following two weeks of growth in normal media (containing light variants of lysine and arginine), heavy lysine and arginine were added in excess as described in Results. Lactacystin was added to one of the sets at this time, resulting in a final concentration of 10μM. 24 hours afterward, cells were harvested and separated side by side by SDS-PAGE. Two lanes in such a gel (stained with Coomassie Blue) are shown. Lanes were then cut into 5 slices as indicated, proteins in each slice were digested, and the resulting peptides from each slice and each treatment were submitted separately to MS analysis, resulting in a list of H/L ratios for each peptide. B) Comparisons of H/L ratios obtained for 1409 proteins in lactacystin and untreated cells. H/L ratios are averages of 2 experiments.
a) Schematic diagram of the domain of ActA involved in actin nucleation.figf2(a) Schematic diagram of the domain of ActA involved in actin nucleation. The signal peptide, the membrane anchor, the proline-rich repeats, the putative actin-binding motif and the Arp2/3 complex-recruiting motif are shown in violet, brown, yellow, pink and light-blue, respectively. The deleted amino acid regions of ΔactA3, 5, 9, 13, 16 and 17 are 129-153, 265-389, 142-153, 90-109, 154-262 and 390-584, respectively. ΔactA14 has R148S substituted for a single amino-acid. ΔactA21 harbours deletions of amino-acids 93-98, 265-389 and the substitution R148S. Right, semiquantitative assessment of actA mutants for bacterial motility, recruitment of the Arp2/3 complex or VASP, and entrapment by autophagy. +, positive; ++, very strong. (b, c) Quantification of the number of GFP-LC3-positive bacteria (b) or actin-accumulating bacteria (c). Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 3 experiments). (d) Colocalization of ubiquitin with ΔactA2. MDCK/pGFP-LC3 cells infected with ΔactA2 bacteria were stained with an anti-Listeria antibody (blue) and anti-ubiquitin antibody (red). Scale bars, 10 μm. Arrowheads indicate GFP-LC3-positive or ubiquitin-positive bacteria. (e) High magnification of framed image in d, and the graph shows the intensity of the fluorescence signal along the arrow. (f) Quantification of the number of GFP-LC3-positive, ubiquitin-positive or GFP-LC3- and ubiquitin-positive bacteria. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 3 experiments). (g) Immunogold electron micrograph of MDCK/pGFP-LC3 cells infected with ΔactA2. Gold particles of 5 nm (arrowheads) indicate GFP-LC3, and gold particles of 10 nm (arrows) indicate ubiquitin. Scale bars, 0.5 μm. (h) Kinetics of the generation of GFP-LC3-positive or ubiquitin-positive bacteria. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 3 experiments). *P 0.001. (i) Quantification of the number of ubiquitin-positive or GFP-LC3-positive bacteria. MDCK/pGFP-LC3 cells infected with wild-type or ΔactA2 bacteria were untreated or treated with 3-MA (10 mM). Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n ≥ 3 experiments). Ub, ubiquitin. WT, wild type.
Figure 4Gene Set Enrichment Analysis comparing the transcriptomes of fibroblasts isolated from pre-tumorigenic ear skin of female HPV8/Act-transgenic mice vs. HPV8/wt-transgenic mice (12-14 weeks old) to published datasets of cancer stroma or CAFs from different types of human cancers and from total Actinic Keratosis (AK) samples. Positive or negative Normalized Enrichment Scores (NES) correspond to enrichment of a given set in genes that are up- or downregulated, respectively, in response to activin A. Data are presented as pseudo-heatmap with NES magnitude color-coded as indicated in the legend; all non-grey comparisons have FDR<0.10. Ingenuity pathway analysis of differentially regulated genes in FACS-isolated fibroblasts from activin A transgenic mice in either a HPV8-negative or HPV8-positive background compared to respective controls, showing selected top activated functions. (Top) Functional enrichment of the most up-regulated genes using the Reactome database showing three unique significantly enriched terms. (Bottom) Filtered list of up-regulated genes based on the Matrisome database showing selected top up-regulated collagens, ECM regulators, and secreted factors. mDia2 (Diap3) expression quantified by RNA sequencing of fibroblasts isolated from ear skin. N=3 pools of 3-6 mice. Primary mouse fibroblasts were treated with activin A or TGF-β1 or vehicle for 6h and analyzed by qRT-PCR (N=3) and Western blot for mDia2 (Diap3) and mDia2 expression, respectively. The GAPDH loading control is identical to the one in Fig 2C, since the same membrane was probed for mDia2, αSMA and GAPDH. Alignment of the sequences in the first intron of mDia2 (Diap3) genes of different species that harbor a SMAD2/3 binding element (SBE) (consensus sequence highlighted in red and italics) (top). Chromatin-immunoprecipitation of lysates from activin A- or TGF-β1-treated fibroblasts using a SMAD2/3 antibody and amplification of the bound DNA with mDia2 primers (bottom). Pre-immune serum (IgG) was used as negative control, and the input chromatin is shown. N=3. Immortalized mouse fibroblasts were transduced with a lentivirus expressing a DOX-inducible dominant-negative ActRIB mutant (dnActRIB) or an empty lentivirus (EV), treated with activin A for 2.5 h and analyzed by Western blot for mDia2 and GAPDH. Representative sections of ear skin tumors formed by SCC13 EV or SCC13 Act cells (clone 2) stained for mDia2 or K14 (green) (top), mDia2 (green) or MECA32/LYVE1 (red, bottom) and counterstained with Hoechst (blue).
Figure 3a. Residues associated with the binding of the UBA domain to the UBC domain were analyzed through sequence alignment of Ubc1 with other E2 enzymes from S. cerevisiae. Y76 and W144 in Ubc1 were predicted to be hot spot residues of the interface, while L143 and L147 were classified as contributing amino acids b. L143E and/or L147E amino acid substitutions were introduced into Ubc1 to disrupt the hydrophobic UBC/UBA domain interface. Single turnover ubiquitination experiments were performed with these Ubc1 variants in presence of K63‑Ub2 with Ub(K48R) at the distal position or monoubiquitin as acceptor. Experiment and initial rate determination c. Ubc1 variants were generated with deletions in the linker region (aa152‑167), which connects the UBA domain and the UBC domain. Single turnover ubiquitination reaction mixes were incubated for 5 min with K63-Ub2 or for 20 min with monoubiquitin as acceptor. Final product formation was analyzed by SDS-PAGE and fluorescence scan. Product fluorescence over total fluorescence (rel. fluo.) was quantified. d. C-terminally blocked M1-linked diubiquitin with or without a K48R amino acid substitution at the distal or proximal position were employed in single turnover experiments with full-length Ubc1 or Ubc1-ΔUBA. Reactions with acceptor K63-Ub2 were carried out controls. Initial rate determination