EMBO/sd-character-level-ner huggingface dataset:
Genetic inhibition of PI3KC2β reduces inflammation 24 hours after stroke.Figure 2WT and C2βD1212A/D1212A mice were subjected to tMCAO followed by 24h of reperfusion. (A) Gene expression of TNFα, IL-1β and IL-6 in the cortex and basal ganglia (B. ganglia). The mRNA levels are given as the fold change normalized to rps29 relative to the corresponding contralateral (healthy) hemisphere. Data represent mean ± SEM (n=8-10 mice per group; *p=0.05; **p<0.01, Unpaired t-test with Welch's correction). Data represent mean ± SEM (n=5-7 mice per group; **p<0.01; Mann-Whitney test). WT and C2βD1212A/D1212A mice were subjected to tMCAO followed by 24h of reperfusion. (B) Protein expression of TNFα, IL-1β and IL-6. Data represent mean ± SEM (n=4 mice per group; *p=0.05; Mann-Whitney test). Data represent mean ± SEM (n=5-7 mice per group; **p<0.01; Mann-Whitney test). WT and C2βD1212A/D1212A mice were subjected to tMCAO followed by 24h of reperfusion. (C) Representative immunohistochemistry of neutrophils (arrowheads) in ischemic hemisphere (scale bar 200 µm) and graph quantification. Data represent mean ± SEM (n=5-7 mice per group; **p<0.01; Mann-Whitney test). D Representative T2-weighted MRI of WT and C2βD1212A/D1212A mice taken 24 hours after clot formation and graph quantification of area stained by P-Selectin using MPIOs in ipsilateral (ischemic) normalize to contralateral (healthy) cortex in percentage. Data represent mean ± SEM (n=10-11 mice per group; *p<0.05; Unpaired t-test with Welch's correction).
Figure 6Upon phage infection, bacteria (dashed-line cells) generate a yet unidentified "danger signal" (red circles) that is received by uninfected neighboring cells. In turn, the latter cells activate SigX that induces the transcription of the dlt operon. The produced Dlt enzymes modulate the phage receptor WTA polymers to reduce phage binding, providing temporary protection against phage attack and limiting phage spread.
Many COP1 substrates and interacting photoreceptors contain VP-peptides that can bind the COP1 WD40 domain.Figure 4A. Table summarizing affinities (Kd, dissociation constant) of VP-containing peptides versus the COP1 WD40 domain (WT) and COP1Lys422Ala as determined by ITC. All values are μM (± standard deviation). B. A sequence comparison of VP-peptide motifs that interact with the COP1 WD40 domain. The VP core is colored in red and the anchor residues in orange. The peptide sequences are numbered on a register where the V in the VP is 0. C. (Top panel) Superposition of the VP-peptide binding modes of the HY5 (green), COL3 (orange), and STO (gray) peptides depicted in ball-and-stick representation when bound to the COP1 WD40 domain as observed in their respective x-ray crystal structures. (Bottom panel) A comparison of the HY5, COL3, STO, CRY1, and UVR8 peptides highlight the chemically diverse anchor residues (orange) and their variant positions. D. ITC assays between the COL3 VP-peptide (left) and CRY1 VP-peptide (right) versus the COP1 WD40 and COP1Lys422Ala, with a table summarizing their corresponding affinities. The following concentrations were typically used (titrant into cell): COL3 - COP1 (2240 μM in 195 μM); COL3 - COP1Lys422Ala (1500 μM in 175 μM); CRY1 - COP1 (750 μM in 75 μM); CRY1 - COP1Lys422Ala (1500 μM in 175 μM). The inset shows the dissociation constant (Kd). The stoichiometries of binding (N) for COL3 - COP1 = 0.75 ± 0.2; COL3 - COP1Lys422Ala = 0.90 ± 0.2; CRY1 - COP1 = 0.99 ± 0.3; CRY1 - COP1Lys422Ala = 0.98 ± 0.1 (All measurements ± standard deviation). E, F. The crystal structure of the (E) COL3 peptide and (F) CRY1 peptide bound to the COP1 WD40 domain. The peptides are depicted in ball-and-stick representation. Selected residues from COP1 are depicted in gray in stick representation.
Deletion of hnRNP L binding site in the BCL2 3'UTR results in increased apoptosis in SU-DHL-6 cells.Figure 7A Schematic of deleted region in CRISPR/Cas9-edited cells. A portion of the 3'UTR at the BCL2 locus is represented, with the last exon as a wide box and the 3'UTR as a narrow box. The light blue region of the 3'UTR represents its first 400 nucleotides, in which the relevant hnRNP L binding sites are located. The "X" indicates the region of the deletion. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. B SU-DHL-4 and SU-DHL-6 cells were treated with varying doses of BCL2 inhibitor. Cell viability was measured after 24 hours of drug treatment using an assay for ATP concentration as a proxy for live cells. Traces were generated from at least 5 independent biological replicates. Points represent mean ± SD at each dose. P value from comparison of dose response between SU-DHL-4 and SU-DHL-6 cells calculated using sum-of-squares F test is given in the graph key. Significance of individual points calculated using two-tailed Student's t-tests is marked using asterisks on the graph. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. C qPCR of selected transcripts in SU-DHL-6 cells under conditions of hnRNP L knockdown or non-targeting control (n = 5 independent experiments). Bars indicate mean ± SD. Significance of hnRNP L KD was determined compared to NT using two-tailed Student's t-tests. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. D Representative amplification of the region of chromosomal deletion in edited cells. PCR products were run on a 1% agarose gel to resolve the amplicons from edited and unedited chromosomes. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. E qPCR of selected transcripts in edited SU-DHL-6 cells (n = 4 independent experiments). Significance was determined as compared to control Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. F Quantification of protein reduction and western blot of lysates from a representative experiment of edited cells. Target protein levels from all biological replicates were normalized to MATRIN3. Each point represents a trial (n = 4). Bars indicate mean ± SD. Significance was determined compared to NT using two-tailed Student's t-tests. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. G FACS-based apoptosis analysis of edited SU-DHL-6. Quantification of relative change in each quadrant is provided, where points represent individual experiments (n = 6 for a given condition). Mean with standard deviation are indicated on the plot, analyzed as in (F). Shown is a representative FACS experiment. Percentage of cells in each gate is indicated. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
Mitochondrial network changes during muscle atrophy.Figure 1(A) Adult muscles were transfected with YFP‐LC3 and with pDsRed2‐Mito. Animals were starved for 24 h or denervated for 7 days. Muscles were exposed and observed in situ using confocal in vivo microscopy. At least five animals per condition have been studied. Scale bar, 20 μm. (B) Adult muscles were transfected with YFP‐LC3 and with pDsRed2‐Mito. Lysosomes were inhibited by treating mice with 50 mg/kg of chloroquine for 7 days. Chloroquine treatment started at the day of denervation and lasted for 7 days, whereas for fasting experiments at the 6th day of chloroquine injection food was removed for 24 h. Muscles were exposed and observed in situ using confocal in vivo microscopy. At least five animals per condition have been studied. Scale bar, 20 μm.
expression (A) Schematic representation of human Fundc1 promoter deletion constructs and 2 putative NRF1 binding sites were marked in blue and red and the corresponding luciferase activity expressed as the activity relative to the 2000 bp promoter.Figure 3(A) Schematic representation of human Fundc1 promoter deletion constructs and 2 putative NRF1 binding sites were marked in blue and red and the corresponding luciferase activity expressed as the activity relative to the 2000 bp promoter. (B) Luciferase activity stimulated by the human Fundc1 promoter. The activity of Fundc1 promoter fragments, either wild type (WT) or with a mutation in the potential NRF1 binding site, was assayed using a dual luciferase reporter system. (C,D) EMSA assay using biotinylated oligonucleotide probes containing the predicted NRF1 binding site or mutant NRF1 binding site in the promoter of Fundc1 (F1 probe or mut F1 probe) ,the confirmed NRF1 binding site in the promoter of VSNL1 (positive probe). The probes were incubated with HeLa nuclear extracts, then run on a gel (C). A super-shifted band (asterisk) is detected in the presence of an anti-NRF1 antibody (D). Labeled probe: biotin labeled wild type Fundc1 probe; Cold probe: wild type Fundc1 probe; Mut probe: NRF1 binding site mutant Fundc1 probe; asterisk indicates the supershifted band generated with antibody to NRF1. (E,F) ChIP assay to analyze the interaction of NRF1 (E) or PGC-1α (F) proteins with the Fundc1 promoter. (G) NRF1-myc was introduced into NRF1 shRNA knockdown cells for 24 h and scramble cells were used as control and samples were collected for western blotting with the indicated antibodies. (H) Scramble and NRF1 shRNA knockdown cells were transfected with vector and PGC-1α-Flag for 24 h and samples were collected for western blotting with the indicated antibodies. (I) ChIP analysis of the interaction of PGC-1α protein with the Fundc1 promoter in scramble or NRF1 shRNA knockdown cells.
Figure 3A U2OS cells were subjected to immunofluorescence staining. Example images of mitotic cells at various stages are shown. Data information: Scale bars, 10 μm. B U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were subjected to immunofluorescence staining. Example images of anaphase cells are shown. Data information: Scale bars, 10 μm. C U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were subjected to immunofluorescence staining with DAPI, and antibodies for TOP2A and H2ApT120 or H3pS10. Example images of interphase cells are shown. The cells in the top line, which is H3pS10-positive, are in early prophase/late G2. Data information: Scale bars, 10 μm. D U2OS-LacO cells transiently expressing EGFP-LacI, EGFP-LacI-Bub1-K (WT or D946N) were subjected to immunofluorescence staining. Arrows point to the transgene loci. Data information: Scale bars, 10 μm. E, F MBP-TOP2A (429-1531)-10xHis was subjected to pulldown by GST, GST-H2A, or GST-H2ApS120, followed by immunoblotting with antibodies for MBP, GST or H2ApT120 (E). The relative pulldown efficiency was determined (F). Means and error bars representing S.D. from three independent experiments are shown (unpaired t-test).
The LIR domain in Atg1 is functionally important in vivo.Figure 3(A) atg1Δatg13Δ cells containing an empty control plasmid, wild‐type or the indicated Atg1 mutants, and Atg13 wild‐type, the Atg13‐FVmutant or an empty plasmid were grown to mid log phase. Processing of endogenous Ape1 was analysed by western blotting, and quantified as described in the legend of Figure 1I. Expression of the different proteins was controlled by immunoblotting with antibodies against Atg1 or Atg13. The asterisk marks a non‐specific band detected by the Atg13 antibody. (B) pho8Δ60pho13Δatg1Δ cells expressing wild‐type (wt), kinase‐dead (kd), Atg1‐VE (VE) or ‐EYE (EYE) mutants or an empty plasmid were grown to mid log phase and starved for 4 h in SD‐N medium. Pho8Δ60‐specific alkaline phosphatase (ALP) activity (nmol/min/mg) was measured in three independent experiments as described in 'Materials and methods', and plotted as relative ALP activity with standard deviation (s.d.) compared to the wild‐type controls. (C) Cells as in (A) expressing GFP‐Atg8 were grown to mid log phase with starvation for 4 h in SD‐N medium. The processing of GFP‐Atg8 was analysed and quantified as in Figure 1I. (D) Exponentially growing atg1Δ and atg1Δatg8Δ strains expressing the PAS marker Ape1‐RFP and either GFP‐tagged wild‐type Atg1 or the Atg1‐VE mutant were examined by fluorescence microscopy. Bar=5 μm. (E) atg1Δ cells containing TAP‐tagged wild‐type or the indicated Atg1‐mutants were grown to mid log phase. Atg1 was immunoprecipitated and its in vitro autophosphorylation activity was measured by autoradiography. The kinase‐dead (kd) Atg1‐mutant K54A serves as a negative control. Figure source data can be found with the Supplementary data.
The minimal Pellino-1-binding region of Smad6 selectively inhibits NF-κB signaling.Figure 2A Schematic representation of truncated mutants of the Smad6 MH2 domain. B A plasmid encoding the truncated mutant composed of Smad6 amino acids 422-441 (Myc-Smad6(422-441)) or wild-type Smad6 MH2 domain (Myc-Smad6-MH2) was co-transfected with the HA-tagged full-length Pellino-1 plasmid into HEK293 cells. Cell lysates were immunoprecipitated (IP) with anti-Myc or anti-HA antibody, and immunoblotted (IB) with anti-HA or anti-Myc antibody, respectively. The vector, CS3MTBXA-6xMyc, was transfected as a negative control. IP, immunoprecipitation; IB, immunoblot; TCL, total cell lysates. Data are representative of at least three independent experiments. C The Myc-Smad6(422-441) plasmid was co-transfected with full-length Flag-IRAK1, Flag-TRAF6, Flag-MyD88, or HA-Pellino-1 plasmid into HEK293 cells. Cell lysates were immunoprecipitated with the indicated antibodies and immunoblotted with anti-Myc antibody. IgG was added as a negative control for IP. Data are representative of at least three independent experiments. D, E The SBE-Luc or 5xNF-κB-Luc reporter plasmids were co-transfected with an empty vector or the Myc-Smad6(422-441) plasmid into CMT-93 cells, respectively. After 24 h, cells were treated with TGF-β1 for 6 h or LPS for 2 h, and luciferase activities were measured and normalized. F The BRE-Luc reporter plasmid was co-transfected with an empty vector or the Myc-Smad6(422-441) plasmid or full-length Smad6 into RAW264.7 cells, respectively. After 24 h, cells were treated with BMP4 for 6 h and luciferase activity was measured and normalized. G A plasmid encoding the truncated mutant composed of Smad6 amino acids 422-441 (Myc-Smad6(422-441)) or wild-type Smad6 MH2 domain (Myc-Smad6-MH2) or full-length Smad6 (Myc-Smad6) was co-transfected with HA-tagged full-length Smad4 or HA-tagged full-length Pellino-1 plasmid into HEK293 cells, respectively. Cell lysates were immunoprecipitated (IP) with anti-Myc and immunoblotted (IB) with anti-HA or anti-Myc antibody, respectively. The vector, pCS3MTBXA-6xMyc, was co-transfected with HA-Smad4 or HA-Pellino-1 as a negative control. Data are representative of at least three independent experiments.
Prazosin inhibits glioblastoma growth in vivo.Figure 2A. Schematic representation of prazosin treatment in vivo. EGFR+/CD133+ GICs directly isolated from primary human glioblastoma samples (GBM44 and GBM5) were transduced with a GFP-luciferase construct and orthotopically implanted into the striatum of NSG mice. Treatment was initiated once the tumors were detected and bioluminescent analyses of tumor growth were performed after 45 days of treatment. B, C. In vivo effect of prazosin treatment (1.5mg/Kg) on glioblastoma growth. Tumors were initiated with GBM44 GICs (B) or GMB5 GICs (C). Left panels: Bioluminescent in vivo images of tumors in mice treated with prazosin (PRZ) or vehicle for 45 days. Middle panels: Quantification of the bioluminescent signals. Fold change in total flux represents the ratio: total flux after treatment/total flux before treatment. *P=0.0002 and *P=0.003 for GBM44 and GBM5 respectively, n=8, two-sided Mann-Whitney U-test. Right panels: Kaplan-Meyer survival curves of mice treated with prazosin (PRZ) or vehicle demonstrating a significant survival benefit of prazosin as compared to vehicle, log-rank Mantel-Cox test. The treatment period is shaded in gray. D. Example of hematoxilin/eosin staining of brain coronal sections from mice sacrificed after 45 days of treatment with prazosin (PRZ) or vehicle. i: tumor infiltration. n: tumor necrosis. Scale bar = 2 mm. GBM44 GICs. E. Left panel: Representative flow cytometry plots depicting the percentage of CD133+ glioblastoma cells (GFP+) isolated from mice treated with prazosin (PRZ) or vehicle. Tumors were initiated with GBM44 GICs. Right panel: Quantification of flow cytometry analyses of CD133 expression by GFP+-tumor cells isolated from xenografts of three prazosin-treated (PRZ) mice and three vehicle-treated mice. *P=0.0003, n=6, two-sided Mann-Whitney U-test. F. Secondary grafting of tumor cells (GBM44-GFP+) isolated from vehicle or prazosin-treated mice bearing primary tumors. Mice injected with glioblastoma cells isolated from prazosin-treated mice developed tumors at a lower frequency (50% versus 100%). Moreover, mice injected with glioblastoma cells isolated from prazosin-treated mice presented a statistically significant survival benefit. Left Panel: Bioluminescent in vivo images of secondary grafts. Middle panel: Quantification of the bioluminescent signals. *P=0.0004, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice bearing secondary graft from glioblastoma cells isolated from vehicle- and prazosin-treated mice. G. Inhibitory effect of low doses of prazosin (0.15 mg/Kg) on glioblastoma growth in vivo. Left panel: Bioluminescent in vivo images of tumors in mice treated with vehicle or prazosin (PRZ) for 45 days. Tumors were initiated with GBM44 GICs (compare with panel B). Middle panel: Quantification of the bioluminescent signals. *P=0.0007, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice demonstrating a significant survival benefit of a treatment with low doses of prazosin, log-rank Mantel-Cox test. The treatment period is shaded in gray.
Characterization of pcMNPs.Figure1TEM image and (B) size distribution of pcMNPs; The average size of nanoparticles is represented as mean ± SD of over 250 individual particles. Zeta potentials of bare MNPs, NH2-MNPs and pcMNPs in water. (D) The magnetic response property of bare MNPs, NH2-MNPs and MBs in 30 s.
Figure 3Graphical explanation of the effect of PHF6 on checkpoint recovery through DNA repair and p53. U2OS cells were transfected with four different siRNA oligonucleotides targeting PHF6, synchronized in G2 and subsequent recovery after 5 Gy of IR and the addition of nocodazole was analyzed by flow cytometry with MPM2 staining PPM1D and βTrCP were used as positive controls. U2OS cells were transfected with four different siRNA oligonucleotides targeting PHF6, synchronized in G2 or cells were lysed and analyzed using western blotting with the indicated antibodies U2OS WT, PHF6 knockout and PHF6 knock out cells that were reconstituted with GFP-PHF6 were synchronized in G2 and recovery was determined after 2 Gy of IR using flow cytometry with pHH3 staining U2OS WT, PHF6 knockout and PHF6 knock out cells that were reconstituted with GFP-PHF6 were synchronized in G2 cells were lysed and analyzed using western blotting with the indicated antibodies U2OS WT and PHF6 knockout cells were irradiated using 3 Gy of IR and cells were fixed at different time points for immunofluorescence. In (F) is presented 53BP1 IRIF intensity. In (G) is shown the number of γH2AX foci per cell. U2OS WT and PHF6 knockout cells were left untreated or irradiated with 3 Gy and processed after 1 h for comet assay analysis. The comet tail moment was analyzed in at least 50 individual cells.
a) Primary cortical neurons (DIV 5) were treated with SFN (10 μM,.f3(a) Primary cortical neurons (DIV 5) were treated with SFN (10 μM, SFN) for 12 or 24 h. (c,d) Bar graphs represent the relative optical density of phosphorylated tau normalized with that of total tau. Data shown are mean±s.e. of three independent experiments and were analysed using Student's t-test. (*P0.05; **P0.01). (b) Stably transfected CN1.4 cortical neurons were maintained in the presence of doxycycline (1 μg ml−1) to induce the expression of tau for 24 h, and subsequently treated with SFN for 12 or 24 h. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using the 12E8 and PHF1 antibodies, respectively. Tau was detected with a polyclonal phospho-independent tau antibody (Tau). The relative molecular masses (kDa) are indicated to the left of each blot. (c,d) Bar graphs represent the relative optical density of phosphorylated tau normalized with that of total tau. Data shown are mean±s.e. of three independent experiments and were analysed using Student's t-test. (*P0.05; **P0.01).
a) EGF-stimulated endocytosis and post-endocytic trafficking.figf7(a) EGF-stimulated endocytosis and post-endocytic trafficking. Uptake of Alexa Fluor 488-EGF (green) by HCT116.Vec (left panel) and HCT116.UVRAG (right panel) cells was followed by confocal microscopy. At the indicated times, cells were fixed and stained with antibodies to LAMP1 (red), and Topro-3 (blue, to stain the nucleus). Magnified images of the insets (fourth row) highlight trafficking of EGF towards LAMP1-positive structures. (b) EGFR degradation. HCT116.Vec, HCT116.UVRAG and HCT116.UVRAGΔN(1-147) cells were treated with EGF (200 ng ml−1) at 37 °C for the indicated times. WCLs were subjected to immunoblotting with an anti-EGFR antibody. EGFR at each time point was quantified and indicated as a percentage relative to that at time zero (right panel; data are mean ± s.e.m., n = 3). (c) DQ-green BSA dequenching assay. Representative confocal microscopy images of DQ-BSA proteolysis (left panel) and flow cytometry analysis (right panel) of RAW264.7 cells expressing an empty vector (RAW264.7.Vec; dotted black line), wild-type UVRAG (red line) or UVRAGΔN(1-147) mutant (green line) loaded with 10 μg ml−1 DQ green-BSA for the indicated times. Background (grey line) represents samples without the BSA load. (d) Effect of UVRAG on endosome fusion. RAW264.7.Vec and RAW264.7.UVRAG cells were pulse-labelled with Oregon green 514-avidin and dextran-568 for 10 min and chased for 30 min. Cells were then labelled with biotin-BSA for 10 min, and processed for confocal microscopy (left panel). In the absence of biotin-BSA, Oregon green514-avidin showed very low fluorescence (first row of confocal image). The percentage of dextran-568-labelled endosomes with visible Oregon green staining (denoted by arrows in the left panel) was calculated from approximately 20 different cells in 2 independent experiments (mean, right panel). Scale bars, 5 μm. The raw data of the immunoblots in b are shown in the Supplementary Information, Fig. S6.
Figure 4A. Side views of the NS-EM structure of the T4SS3-10 (left) and T4SS3-10+D4 (right) complexes. The arches, the TrwK/VirB4 barrels, and the TrwB/VirD4 density are shown in green, yellow, and blue, respectively. The boundaries of the 3-tiers structure of the TrwK/VirB4 barrel are indicated in the middle of the panels and each tier is labelled as lower, middle, and upper, respectively.  B. Side view of the T4SS3-10+D4 structure (upper panel) and derived schematic representation (lower panel). Overall dimensions are indicated C. Same as in B except that the side-view shown is rotated 90 degrees along a vertical axis compared to the view shown in panel B. Overall dimension is indicated. D. Bottom view of the NS-EM structure of the T4SS3-10 (upper panel) and the corresponding schematic representation (lower panel). Angle between the axes connecting the TrwK/VirB4 hexamer pair and the TrwB/VirD4 dimer pair is shown.
Model.Figure 7ModelSchematic depiction of the localization and topology of the three multipass membrane proteins identified as regulators of endosome-to-Golgi retrieval. Features such as the YxxΦ motifs in ZDHHC5 are indicated