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Munc18-1 knockout neurons expressing G544D Munc18-1 were plated on a microelectrode array before addition of compounds (0 h) or 48 hours after vehicle (DMSO) or compound addition.
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C. At day 21, cells were stimulated with 1 µM prostratin for 24 h and HIV transcripts were quantified by RT-qPCR. Reduction in viral stimulation in Vs treated samples are represented as percentage values. ND - non determined. D. Aggregate plot for 3 patients from data (C).
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(D) Stereological analysis of cortical area covered by Congo Red-positive plaques in APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice (left) and representative images (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, **p=0.0070.
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A Western blot analysis of SDS-PAGE of MGM132-treated 143B APOPT1HA cells. The graph represents the densitometric quantification of the signals for the precursor and mature protein
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(A) Real-time quantitative PCR of the indicated genes (M1-like and M2-like macrophages markers) of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) compared with vehicle after normalization for housekeeping gene TBP (n=3 tumors), (*P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle (PLX4720: m-Arg1 P = 5.65E-06) (bevacizumab: m-Ccl5 P = 0.025) (COMBO: m-Ccl5 P = 0.0070; m-Cd40 P = 0.00099; m-Cxcl10 P = 0.043; m-Cxcl9 P = 0.046; m-Il1b P = 0.0367; m-Stat1 P = 0.049; m-Tlr2 = 0.00182; m-Arg-1 P = 0.0013).
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Growth curves of the MCF7 (D) or T47D cells (E) upon HNRNPC knock-down but with the interferon response blocked with the IFNβ antibody. Each sample has 3 replicates. Error bars represent mean ± SD.
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C. MicroRNAs having at least 5 reads in 50% of the samples were considered for downstream co-expression analysis. Co-expression analysis revealed 3 microRNA clusters that were significantly linked to weighted cognitive performance. Number of microRNAs in each module is given in parentheses next to module name. Co-expression modules are represented in rows, while each column refers to a phenotypic trait. Each cell contains the corresponding correlation coefficient and p-value (denoted inside parentheses). Color code represents Pearson´s correlation. Expressions of the modules are not correlated with sex, number of years at school or status of total education.
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(a) HeLa cells transfected for 24 h with GFP-Atg16L1 (green) and CFP-LC3 (blue) were incubated with Alexa Fluor-555-conjugated cholera toxin subunit B (red) for 15 min at 4 °C (allowing toxin to bind to the plasma membrane). Then cells were incubated at 37 °C (allowing internalization of cholera toxin) for 10 min and fixed for confocal analysis. Vesicles positive for both Atg16L1 and cholera toxin are yellow (also see high-magnification images at the right). Note that the small Atg16L1 vesicles co-localizing with cholera toxin are negative for LC3 (as marked with yellow arrows), and the Atg16L1 vesicles co-localizing with both cholera toxin and LC3 (marked with blue arrows) are shown in the magnified panels at the right. Scale bar, 10 μm.
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14‐day‐old seedlings grown on ms plates were used for RNA extraction. Next, 1 μg of RNA from each sample was treated with XRN1 (NEB) or mock treated before RT‐qPCR. Data were normalized to ACT2, and transcript levels were compared between XRN1 and mock‐treated RNA for each genotype. Standard error of the mean is indicated by error bars (n = 4). Statistical significance between the mean values was determined by ANOVA followed by Fisher's LSD test.
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F Lung weight of mice from (E). Kruskal-Wallis test followed by post-hoc analysis with Dunn's test. P-values were adjusted with Bonferroni's correction to account for multiple comparisons. Only the groups 100% WT and 0% WT resulted to be significantly different (*p=0.0254). Median ± IQR.
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B-C. The Rab25 expression signature identifies patients with a poor prognosis. Ovarian cancer patients were classified as either mirroring the Rab25-associated gene expression signature (Rab25 signature) or not (non-Rab25 signature) using linear discriminant analysis in BRB tools, in two independent published ovarian datasets. Progression free survival curvesfor patients with advanced disease (Stage II to IV) are shown. Univariate analyses were plotted using Kaplan-Meier method and Coxplots used for multivariate analyses (co-variates included stage, grade, histology and residual disease).B. Tothill et al, 2008.C. Dressman et al, 2007.
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D HeLa WT or FBXO2-KO cells transfected with mCherry-Gal3 (magenta) were infected with WT GAS for 2 or 4 h, and percentages of Gal3-positive GAS-containing cells were quantified.
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B ARBE reporter constructs were transiently transfected into LNCaP cells. Cells were stimulated with DHT (100 nM), a synthetic androgen R1881 (10 nM), or vehicle for 16-24 h, and luciferase activities were measured. The results were presented as the mean ± SD of the quadruplicate transfections. The genomic positions of ARBEs are also shown.
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(I) The schematic diagram shows that cystine deficiency-induced AMPK activation is mediated by CaMKK2, which is recruited by CARS and the AMPKγ2 complex.
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(H) Quadriceps and gastrocnemius wet weight of 6 months old mice was similar between WTFlx and KO-NDUFS3 mice. Quadriceps weight was significantly increased in KO-NDUFS3 mice when compared to KO-GFP injected. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. ns- not significant. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.
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Percentage of cycling Ter119- Kit+ CD41+ CD16/32+ EMPs and Ter119- Kit+ CD41- CD71+ CD44+ early erythroid (Early E) cells in the E11.5 FL, determined by flow cytometric analysis of BrdU and 7-AAD incorporation. Data are the mean (±SD) of 6 wild type and 5 Sl/Sl FLs analyzed individually over 3 independent experiments. Tail somite range: 12-17 (+/+); 12-17 (Sl/Sl).
|
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(A) Structure of the TRIM32 RING dimer in ribbon representation with each RING monomer coloured in cyan and blue and the Zn2+ ions as grey spheres.
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F: Quantification of the glomerulosclerosis score at time of (M0), or 12 months after (M12) kidney transplantation from elderly donors in patients displaying a negative (PAI-1-, n = 17) or positive (PAI-1+, n = 13) glomerular PAI-1 staining at time of transplantation (M0). Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test
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(A) Female athymic mice were injected subcutaneously with either CP20 GFP-miR-CTL or CP20- GFP-miR-195 cells. Mice injected with the miR-195 expressing cells (miR-195) had significantly smaller tumor volumes than control mice (miR-CTL). Data are mean ± S.D, n=10. *P<0.05, Student's t-test.
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C Real-time RT-PCR analysis of primary transcripts of ER-GATA-1 target genes (n = 4, mean +/- SE).D Fold changes of primary transcripts with 5-ALA treatment in comparison with no treatment in b-estradiol-treated cells (n = 4, mean +/- SE). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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(B) Western blot of adult males and females showing disappearance of Yki1 isoform in the two yki2-only fly lines.
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Cyclin E-expressing BJ cells grown for 7 days with and without folate.Immunoblotting with anti-phosphorylated ATM and anti-phosphorylated CHK1 antibodies. Anti-β-catenin and anti-actin antibodies were used as loading controls.Protein level quantification.
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Measurements of SIV-VSV-luc infectivity in FRhK4 (J) cells depleted of CLIP170. Statistical significance was determined by t test. *** = p<0.001. Data are mean values from two independent experiments +/- SEM.
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D. Schematic of a molecular chain of n events triggered by stress and resulting in measurable fluorescence, according to the statistical kinetics model applied here. The gray arrows indicate possible deviations from a linear chain of events, like feedback, reversibility, or branching, which are also captured in the model.
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B. Activation of ßcatenin signaling by F4L5.13 or recombinant NDP (30nM each) in HEK293T cells transfected with FZD4 and/or LRP5. Values represent fold-activation of LEF/TCF reporter gene. Data are presented as mean ± SEM, n=3.
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Model for the roles of Rubicon and UVRAG in the maturation of autophagosomes in HCV-infected cells.In the normal autophagic pathway, UVRAG, in complex with Beclin-1, p150 and Vps34, facilitates the fusion between autophagosomes and lysosomes to form autolysosomes. The induction of Rubicon by HCV in the early stage of infection inhibits the UVRAG activity and the fusion between autophagosomes and lysosomes. This leads to the accumulation of autophagosomes, which enhance HCV RNA replication. The induction of UVRAG in the late stage of HCV infection overcomes the inhibitory effect of Rubicon and results in the maturation of autophagosomes. In the model illustrated, the effect of HCV on the initiation of autophagy is not addressed.
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(D) Flow cytometric assessment of infiltrating total leukocytes (CD45+), granulocytes (CD45+ Ly6G+ Ly6C-), and inflammatory monocytes (CD45+ Ly6G- Ly6C+) recovered in the synoival fluid of the knee joints of mice treated as in (A). Data is displayed as floating bars with the max/min values and mean (thicker band). Biological replicates are: PBS + Vehicle, n=4; PBS + VHHmASC, n=3; mBSA + Vehicle, n=6; mBSA + VHHmASC, n=6; mBSA + IL-1RA, n=5.
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(a) Flow cytometry. Untreated Jurkat T cells, cells treated with etoposide continuously (0.25 μM), or transduced with lentivirus ATG5 or GFP were cultured for 48 h, fixed, permeabilized and exposed to antiphospho-H2AX (Ser139) antibody, then stained with antimouse-APC-conjugated antibody, and analysed using a flow cytometer.
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B. T6SS secretion assay of A. tumefaciens wild type C58, ΔtssL, Δtdei, and Δ3TIs harboring the indicated plasmids.
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(E) The combined average mRNA levels of ND1, COII, COIII, Cyt b and ATP6/8 in HeLa cells treated with either NT or siLETM1 (siR1, siR2 or siR3). Data are mean values ± SEM, n=3 independent experiments
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A. Gene regulatory network underlying mESC differentiation to the three germ layers. Gray node shades indicates the significance of the motif activity, edge thickness indicates the strength of the interaction.
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Shown are the schematics and responses for th oxygen sensors Horizontal error bars in the PfnrF8 response reflect one standard deviation of three DO measurements Representative cytometry florescence distributions for Figure 1g
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B Representative images of back skin of the DMBA-treated Hgf-Cdk4R24CXiapfl/fl and Hgf-Cdk4R24CXiapKO mice at 8 weeks of age.
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A Schematic domain organization of S. aureus Bap. S, signal peptide (aa 1-44); A, region A (aa 45-360); B, region B (aa 361-818); SP, spacer region (aa 819-947); C, repeat region (aa 948-2139); D, serine-aspartate repeats region (aa 2148-2208); W, cell wall anchor region. The aggregation-prone regions of BSP consists of 5 modules: 1 (magenta), 2 (orange), 3 (green), 4 (blue) and 5 (cyan) based on its crystal structure.
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Immunofluorescence analysis of total TDP-43 and phosphorylated TDP-43 (p-TDP-43) in sagittal brain sections by confocal microscopy. Representative images of one confocal layer in the midbrain. Zoom in on TDP-43 aggregation in double knockout mouse brain sections of the midbrain. Scale bar indicates 25 µm. (Animals 4.5 months of age).
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(C) Quantification of Smc5 sumoylated species in pull downs from the indicated smc5-KE mutants, relative to wild-type controls from at least three independent experiments. Boxes, 25-75% data range; whiskers, total data range; black bar, median; grey cross, mean. ****P < 0.0001; ***P<0.001; **P < 0.01; *P<0.05. 1-way ANOVA, n = number of samples analyzed
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Simulated kymograph of a more polarized cell with broader PI(3,4) P2 enriched region compared (A). Slower vesicle arrival time and reduced decay rate for PI(3,4) P2 was assumed in comparison to parameters used for the simulation in (A).
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F. Confocal images of control (1st column) and NPC1I1061T fibroblasts (2nd column) expressing mCherry-P4M or control fibroblasts expressing P4M-mCherry and GFP-DHHC3 (3rd column).
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D. Analysis of significant GO terms for genes that were downregulated by at least 2-fold in the MEF2C-knockdown cells compared with control cells at day 5 of neural differentiation (hypergeometric test, P < 0.05).
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(G) Changes in the deuterium incorporation of c10orf76 in the presence of PI4KB. H/D exchange reactions displayed as the sum of the difference in HDX in the number of deuterons for c10orf76 (400 nM) in the presence of PI4KB (400 nM) at all time points (3s at 1 oC; 3s, 30s, and 300s at 23 oC) analyzed. Red boxes highlight regions that showed significant changes (>7% decrease in exchange, >0.5 Da difference, and unpaired two-tailed student t-test p<0.05). Error bars represent standard deviation of independent technical replicates (n=3).
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Labeling for CD31 in retinas of P4 and P7 RhojWT/WT and RhojGFP/GFP mice. Scale bars, 500 µm.
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A Relative activity of NF-kB luciferase reporter was measured in A549 cells transfected with plasmids encoding PB1-F2. The mRNA levels of pro-inflammatory cytokines were determined by semi-quantitative RT-PCR. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).
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Representative photomicrographs of TDP-43 (green) and NeuN (red) immunohistochemistry in hippocampal CA1 of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Arrows denote areas of depleted TDP-43 immunoreactivity in the mutant.
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B, C. SCID male mice with PDX tumors were treated with vehicle (40% polyethylene glycol), JQ1 (50 mg/kg), CPI-637 (30 mg/kg), combination of JQ1 (50 mg/kg) and CPI-637 (30 mg/kg) or NEO2734 (30 mg/kg) five days a week for three consecutive weeks. Tumors isolated from mice at day 21 of drug treatment were photographed (B) and tumor growth are shown in (C). All data shown are means ± SEM. The P value comparing the tumor volume at day 21 post- treatment was calculated by the unpaired two-tailed Student's t-test; *** P < 0.001.
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E and F. Western blot results of UCP1 protein in iBAT from WT and TRPV2KO mice before (25oC) and after 3 days cold (4oC) exposure (E). Comparison of UCP1 protein levels before and after cold exposure in iBAT from WT and TRPV2KO mice (F). Mean ± SEM, n = 6; * P < 0.05 vs. 25oC group; # P < 0.05 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.
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L SDD-AGE analysis of MAVS aggregates in HEK293T cells transfected HA-MAVS together with WT Myc-SIRT5 or its enzyme-deficient mutant (Myc-SIRT5-H158Y) after infected with SeV for 12h. SDS-PAGE immunoblotting was used as a loading control.
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Cells were treated with control (CTRL) or NDP52 siRNA, then mock infected (M) or infected (C) for 24 h. Cytoplasmic and nuclear fractions were isolated and immunoblotted for nsP2 and NDP52. The amounts of the cytoplasmic and nuclear fractions of nsP2 were quantified by densitometry and expressed as the ratio of nuclear to cytoplasmic fraction of nsP2 (F).
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(G) Western blot analyses of the degradation of p65 protein after the overexpression of Myc-CDC20. hBMSCs were transfected with Vector and Myc-CDC20 plasmids for 36 h, cells were treated with or without 10 μM MG132 for 6 h before collected.
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(D) Dot plot of selected HP1 high-confidence interactors identified by BioID. The node size displays the relative abundance of a given prey across the 6 conditions analyzed, the node color represents the absolute spectral count sum (capped at 50 for display purposes), and the node edge color corresponds to the Bayesian False Discovery Rate (BFDR). Proteins were selected based on a SAINT score of > 0.95, BFDR of < 0.05, and ≥ 10 peptide count.
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(C-F) Seahorse XF extracellular flux analysis performed on cells 24h after treatment. Sequential injections of oligomycin (Oligo.), FCCP, and a combination of rotenone and antimycin A (Rot. + AmA) were performed to assess mitochondrial fitness. Flux was normalized to relative protein units (RPU) measured after the run with an SRB assay. Data is representative of three independent experiment (n = 3). Error bars represent the standard deviation of biological replicates (SD) (F) Measurements of uncoupled (oligomycin-resistant) and coupled (oligomycin-sensitive) respiration. Coupling efficiency was calculated as the percentage of basal respiration associated with coupled respiration. Data information: Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls
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(F) Binding between FTCD and p47 and between p47 and p97 is necessary for mitochondria aggregation mediated by FTCDwt-HA-MAO. The HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were transfected with mammalian expression constructs of siRNA-insensitive Flag-tagged p47wt/mutants at the same time as the treatment of p47 siRNA, and cultured for 24 hrs. The cells were further cultured in DOX-free medium for 48 hrs for the induction of FTCD-HA-MAO. After fixation, the cells were visualized with a monoclonal antibody to mitochondria and polyclonal antibodies to HA and Flag. Panels a-l display representative images. Scale bar = 10 μm. (G) Binding between FTCD and p97 is necessary for mitochondria aggregation mediated by FTCDwt-HA-MAO. The HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were transfected with the mammalian expression construct of siRNA-insensitive Flag-tagged p97wt/mutant at the same time as treatment with p97 siRNA. The following procedures were the same as in (F). Panels a-i display representative images. Scale bar = 10 μm. (H) The results of quantification of (F) and (G). Results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. Asterisks indicate a significant difference at P<0.01 compared with siRNA treatment alone ('none') and compared with mutant expression (Bonferroni method).
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B) Western blot confirming the absence of FUS in the two selected clones. Extracts from wild-type (wt) and FUS knockout SH-SY5Y cells (clones A4 and A5) were subjected to SDS-PAGE, transferred to a nitrocellulose membrane and FUS (green) and tyrosine tubulin (red; loading control) were detected using respective primary and secondary antibodies.
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HepG2 cells, HAT1 KO HepG2 cells, and HAT1 KO HepG2 cells reconstituted with either wild type HAT1 or mutant HAT1 (T188A) were subcutaneously injected into athymic nude mice (N=6). Western blot analysis was performed with the indicated antibodies. Representative images of triplicate experiments are shown.
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(A) Principal component analysis of DNA methylation data at 17,937,401 CpGs for CD4+ naïve T cells (gray), WT Treg cells (blue) and Tet2/3 DKO Treg cells (red). The percentage of variation explained by each component is shown in parentheses.
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C Detection of MAPKKK5-GFP and PBL27-HA transiently expressed in Nb leaves. Total proteins were purified from Nb leaves at 38 hpi.
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(A) Flg22-induced ROS burst in Gmlmm1-1, Gmlmm1-2 and Williams 82. The indicated leaves (aged 2 weeks) were subjected to flg22-induced ROS examination, and the peak relative luminescence unit (RLU) value was recorded (Mean ± SEM, n ≥ 12, n represents sample number). Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.
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B. Q-RT-PCR analysis of netrin-1 and -3 expression, in lung cancer cell Lines (Error bars indicate SD, n=3).
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C Representative image of cleaved Caspase 3 (Cas-3, upper panel) and TUNEL (lower panel) staining in coronal sections from Kcc2lox/lox cortex at E16.5 electroporated with EGFP or EGFP+Cre are shown. Arrowheads point to neurons expressing Cas-3 (upper panel) and TUNEL (lower panel). DAPI staining (blue) marks cell nuclei. CP, cortical plate; VZ, ventricular zone; SVZ, subventricular zone; Sp, subplate; IZ, intermediate zone. Large scale bar: 50 µm, small scale bar: 20 µm. D Quantification of the percentage of EGFP+ neurons expressing apoptotic markers at E16.5 from embryos electroporated with EGFP ± Cre. Statistical significance was determined using Mann-Whitney U test (Cas-3); and two-tailed t test (TUNEL), ***P < 0.001. Data are presented as mean ± S.E.M., n (-Cre) = 6 embryos; n (+Cre) = 6 embryos. E The number of EGFP+Cre neurons as a percentage of neurons expressing EGFP alone. Statistical significance was determined using a two-tailed Student's t test. Data are presented as mean ± S.E.M., n = 6 embryos.
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(a) HeLa cells transfected for 24 h with GFP-Atg16L1 (green) were incubated with CellMask Orange plasma membrane stain for 5 min at 37 °C, after which they were imaged immediately (in an incubated chamber at 37 °C). A time series after fusion of a CellMask Orange vesicle (red) with a GFP-Atg16L1 vesicle (green) is shown. Scale bar, 5 μm. A higher-magnification image showing co-localization (yellow) of GFP-Atg16L1 vesicle (green) with CellMask Orange-positive vesicle (red), indicated by arrows, is also shown in the top and bottom panels.
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C. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with flow cytometry-quantified MH-S AM-EVs at specified EV:cell ratios.
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(B) Amino‐acid region in TLR2 that includes the motif. Highlighting indicates residues identified by Prosite as part of the motif.
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D) Proteomic analysis of Nhp2 interactors. Highlighted in red are the known members of the H/ACA pseudouridylation complex. The red dashed line indicates significance cutoff
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B) proteomic analyses of different time points in primary CD4+ T-cells infected in vitro with HIV-1 or mock infected. Data were analyzed by Fisher test (number of donors =3 biological replicates). Boxplots in depict median and 25-75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR (inter-quartile range) of the hinge. Data beyond the end of the whiskers are outliers and plotted as points. For each time point, dots below boxplots illustrate the pathway enrichment analysis of proteins up-regulated in infected vs matched mock infected controls. Dots are color-coded based on the enrichment q-values and their size indicates the fraction of differentially expressed proteins in the pathway; grey dots are not statistically significant.
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HeLa and cyclin F Knock-Out (CCNF K/O) cells were treated with Kinase ChemoGenomic Set (KSGS) in 384 well format. After 72 Hours (h) viability was measured using rezasurin. Flowchart representation (left). Robust Z-score difference is plotted for data acquired at 1 μM concentration (right).
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(D, E and F) Polyphenols inhibit the phosphatase activity of RhpS. The GST-RhpR was phosphorylated by adding 5-μM PSPPH_3550c protein as well as 10-μCi [γ-32P]-ATP for 10 minutes. The RhpS-FL protein was treated in the presence of different concentrations of TA (D), PGG (E), or EGCG (F) for 10 min. Then, the full-length RhpS protein was added into the mixture phosphorylated GST-RhpR for 1 hour before stopping the reaction. The phosphorylated proteins were separated by 12% SDS-PAGE. After electrophoresis, the SDS-PAGE gels were dried with gel-dryer and then exposed to a medical X-ray film overnight. This experiment was repeated twice, yielding similar results. Data information: the reaction was stopped by adding 5 × loading buffer before SDS-PAGE. Abbreviation is as follows: CBB, coomassie bright blue stain; CFU, colony-forming unit.
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(F) RNA isolated from kidneys treated with doxorubicin or abemaciclib and mRNA encoding indicated p53-associated SASP genes quantified by qRT-PCR (n=6 mice/group). (G) RNA was isolated from kidneys of p16-3MR mice treated with abemaciclib (50 mg/kg, 7 consecutive days) ± pifithrin-α (2 mg/kg, 7 consecutive days), and mRNA encoding p53 associated SASP genes was quantified by qRT-PCR and normalized on tubulin (n=5 mice/group). ( Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.
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A FRAP images of CAF-1 bodies to indicate the internal diffusion property. GFP-tagged CHAF1A was overexpressed in HEK293T cells. Twenty-four hours later, live cells were proceeded to time series imaging. One of the CAF-1 bodies was bleached with intense laser pulse. Images of live cell were captured every 4 seconds. One of the unbleached CAF-1 bodies was used as control. Data represented eight time points. At least three cells of each sample were treated with intense laser pulse to obtain bleached CAF-1 bodies.
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(A) Normalised concentration values of 16 amino acids in cells grown in EMM without (grey) or with (blue) antimycin A. For each condition, four independent samples were analysed. Concentrations are presented relative to the mean value for the given amino acid in EMM medium. *Ornithine (Orn) is abbreviated as O. Boxplots were created using R boxplot command with default settings. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.
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(I) Similar experiments as in (C) were performed in W1118 and TBPH-/- adult flies. The mRNA levels of lysosomal and autophagic genes were quantified and normalized relative to GAPDH. The data from three independent experiments are presented as means ± S.E.M., ns, not significantly different; **, p<0.01, one-way ANOVA.Total RNA was prepared from W1118 and TBPH-/- larvae and subjected to qRT-PCR analysis. The level of TBPH and raptor were quantified and normalized relative to RPL32. Data from three independent experiments represented as means ± S.E.M., **, p<0.01, one-way ANOVA.
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Cell surface biotinylated LDLR protein levels in HUVECs after 20 min of VEGF-B167 or VEGF-B186 stimulation. Data presented as mean ± SEM from three independent experiments. Western blot shown below. Statistical evaluation using t-test, p-value: * <0.05 (compared to control).
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(A) Alternative splicing of sams-3, sams-4 and sams-5 in smg-2 (yb979) and smg-2 (yb979); sams-5 (gk147); sams-1 (ok2946) mutants. Synchronized L1 larvae of each strain were incubated in S-complete medium alone (lanes 1 and 5), with OP50 (OD600=10.0) (lanes 2 and 6) and with OP50 supplemented with 25 mM L-Met (lanes 3 and 7) or 25 mM cycloleucine (cLeu) (lanes 4 and 8) for 3 hours at 20°C. The splicing patterns were analyzed and presented as in Figure 2B (n=3).
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(A) Western blot analysis PABPDHFR cells depleted of LSM1 by siRNA-mediated knockdown. Transfected cells were subsequently cultured in the presence or absence of TMP to maintain or deplete endogenous PABP.
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B Two or six (-97m3) representative primary transformants expressing GUS under the given PAP8 promoter version; FC+, the corresponding promoters were tested positive in functional complementation of the mutant pap8-1. Scale bar equals 3 mm.
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E Representative human brain metastases showing different intensities or scores for HSP90. Scale bar: 50 µm. F Quantification of HSP90 in human brain metastases. 59 out of 60 (98%) showed positive staining of HSP90 in the tumor, 15 (25%) scored with 3 (strong), 36 (60%) with 2 (moderate) and 8 (13%) with 1 (weak) according to the signal intensity of HSP90 in the cytoplasm of cancer cells.
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U87, T98G and LN229 cells were treated with 1 µM ABT263, 20 µM LXR623 or the combination of both for 72h. Mewo melanoma and HCT116 colon carcinoma cells were treated with 1 µM ABT263, 10 µM LXR623 or the combination of both for 72h (MeWo) or for 48h (HCT116). Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.
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E Decreased paired paired-pulse facilitation (PPF) at hippocampal CA3-CA1 synapses treated with Roscovitine alone (Ros, 10 µM) or VPS34IN1 (3 µM) plus Roscovitine. Representative traces of fEPSPs with a 20 ms interpulse interval is shown above. The ratio of the slope of the second to the first response over a range of interstimulus intervals (10-500 ms) is plotted below. The data show decreased facilitation of the second response in Roscovitine or VPS34IN1 plus Roscovitine-treated acute hippocampal slices, unlike VPS34IN1 alone . N = 6 slices per condition from 6 mice; Two-way RM ANOVA. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001
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(A) Colocalized RGC inputs and PSVue signals in P6 control and CKO. White circles indicate PS+ RGC inputs. Arrows pointing to the RGC inputs enlarged in the insets. (B) The percentage of PS+ RGC inputs in total inputs in CKO and controls.
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] |
A Scheme of H1-GFPFRAP in heterochromatin foci (with HP1a-mCherry) and euchromatin (without HP1a-mCherry). The relative FRAP curves of euchromatin (Eu) and heterochromatin (Het) are shown divided into two parts-the mobile fraction (MF) and the immobile fraction (IF) after bleaching recovery. (ROI, region of interest)
|
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(H) Merlin-depleted LN229 cells stably transduced with wild-type (WT) Merlin or its K396R mutant were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.
|
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C ) Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with the indicated inhibitors: CDC7 inhibitor (PHA-767491) and/or ATM inhibitor (Ku60019). At least 300 replication structures were counted per treatment. Error bars represent SEM. Statistical analysis using two- tailed unpaired t-test (*p < 0.05).
|
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(B-E) Atg16L1-Δ/Δ MEFs stably expressing the indicated constructs were infected with Salmonella (MOI = 10) for 1 h and then analyzed by immunocytochemistry for LC3 (B) or Atg16L1 (D). Bar, 5 µm. The percentage of LC3- or Atg16L1-positive Salmonella per Ub-positive Salmonella was enumerated by fluorescence microscopy (C and E). At least 50 bacteria were counted. The average ± SD is shown for three independent experiments.
|
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One-cell stage embryos were injected animally with mRNAs encoding the noggin (5/20 pg) and/or meis3 (0.5 ng) proteins. AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 17. Total RNA was isolated from seven control embryos (lane 2), and from eighteen ACs from each group (lanes 3-8). Neural AP markers were examined by sqRT-PCR: forebrain (xanf1, otx2), hindbrain (krox20, hoxb1, hoxb3), spinal cord (hoxb9, cdx1-4) and panneural (soxd). Ef1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).
|
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Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(A) Spironolactone reduces ERBB4 levels. T-47D cells were stimulated with 10 ng/ml EGFld, 10 µM lapatinib and 10 µM spironolactone for 5 min as indicated. Cell lysates were probed for ERBB4 phosphorylation levels at Tyr1056 and Tyr1284.
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Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control actin). In , the bars represent the mean±sd of band intensities relative to the actin loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.
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B Lysates of 293T cells transfected with plasmid expressing Flag-Beclin-1 and HA-tagged ubiquitin (HA-Ub (wild type), K6-linked-Ub, K11-linked-Ub, K27-linked-Ub, K29-linked-Ub, K33-linked-Ub, K48-linked-Ub or K63-linked-Ub), together with the empty vector or expression vector of Myc-USP19 and treated with MG132 (10 μM) for 3 h were immunoprecipitated with anti-Flag and immunoblotted with anti-HA.
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D Cellular localization of Zn2+ (blue) in Mx and MdCAX3-suppressed Mx roots under Fe deficient conditions. Arrowhead indicates the cytoplasm. Scale Bars: 20 μm. E Fluorescence intensity detection at the position indicated by the long and narrow arrow in the Fig 6D.
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Volcano plot highlighting up-regulated (red) and down-regulated (blue) genes based on RNA-seq data from GBM#P3 cells after 24OHC treatment. LXR targets, SREBP targets and stemness transcription factors are specifically included.
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D The S622A mutation strongly reduces phosphorylation by PBL27. Each of phosphorylation sites was substituted by alanine, and subjected to the in vitro phosphorylation assays.
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(d,e) CD39, Atg5 and as a control β-actin protein expression in isolated KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes treated with the (d) HIF1α inhibitor 3-(2-(4-adamantan-1-yl-phenoxy)-acetylamino)-4-hydroxybenzoic acid methyl ester or left untreated and (e) the Nrf2 inhibitor Brusatol (100 nM) or the ROS scavenger N-acetyl-L-cysteine (5 mM). Protein lysates were analysed on day 4 by western blot. Quantifications of the CD39/β-actin ratios are shown for each lane. The inhibitors were present during the entire 4 day culture period.
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(C) Viral RNA content as determined in various organs at 3 dpi (n = 6/group) by use of conventional E-specific or sub-genomic Esg-specific qRT-PCR. Red lines indicate the qRT-PCR detection limits. Data information: Statistical significance was evaluated by Mann-Whitney test (* = p<0.05, ** = p<0.01).
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A Top, ASOs mapped onto the mouse ApoER2 exon 19 gene region. Bottom, RT-PCR of RNA isolated from mouse cells transfected with different ASOs. Quantification of percent exon 19 inclusion is shown below the gel image.
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B. Intracellular survival of EGD-(pADc-GFP), EGD-(pADc-inlK), ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in RAW 267.4 macrophages. Statistical analyses were performed on the results of 3 independent experiments using the Student t test. P values of <0.05 were considered statistically different and are labeled here as *.
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(a) Ulk1 Ser 757 is required for mTORC1 to regulate the interaction of Ulk1 with AMPK in vivo. CBP/SBP tagged Ulk1 (wild type or S757C) was co-transfected with HA-AMPKα and Rheb into HEK293 cells as indicated. Ulk1 was purified by streptavidin beads and the co-precipitatedHA- AMPKα was examined by western blot (Rapa, 50 nM rapamycin treatment for 1 h before cell lysis).
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The upper part of the table shows the PWMs over-represented (p-values determined by a two-tailed Welch's t-test are shown in red) in Lo-G PDAC cell lines relative to both enhancers specific of Hi-G PDACs (p vs. Hi-G) and the FANTOM 5 collection of human enhancers (p vs. hs enh) (Andersson et al, 2014). The frequency (f) of these PWMs in the enhancers specific of Lo-G and Hi-G PDACs is also shown. The cognate TFs assigned to these over-represented PWMs are indicated in bold if over-expressed in Lo-G PDACs. The bottom part of the table shows the result of the statistical over-representation analysis for those TFs expressed selectively in Lo-G PDACs but whose PWMs were not over-represented in this analysis (p >1E-5 determined by two-tailed Welch's t-test).
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(F) Mean cfu of Pto DC3000 2 days after syringe inoculation and pre-treatment with 1 μM GLV2; n=20 biological replicates from 5 pooled experiments -/+ SD (Students T-test, *p <0.05).
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(D) HeLa cells KO for LCS were transfected with indicated LCS RUSH constructs for 16 hours, and expressed enzymes were retained in the ER (-biotin) or placed in the Golgi by addition of biotin (40 μM) (+biotin) and SL output monitored by [3H] - sphingosine pulse-chase assay. The ratio of GM/Gb is represented.
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(A) The weighted mass average of WAVE1 PRD (15 μM) determined by sedimentation equilibrium ultracentrifugation with LatB-stabilized monomers (15 μM) and various profilin concentrations as indicated. The lower dashed line indicates the expected mass of WAVE1 PRD; the 5-7 kDa upshift is due to non-specific actin binding (WAVE1 PRD[null], black symbols). The upper dashed line mark the equilibrium mass observed for 15 μM WAVE1 PRD[B] in the presence of 90 μM profilin alone.
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F Endothelin-1 was increased in lung tissue after siISCU delivery under normoxic conditions (N = 5/group), **P = 0.0027.
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J Expression level of the mature NPF1 gene and pre-NPF1 in 293T cells transfected with pre-NPF1 and mutated pre-NPF1 (n=5 biological replicates). NC means negative control. The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value. Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).
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] |
d, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 6 h and immunostained for LAMP1. e, Quantification of results from d expressed relative to control BMDMs (n = 3 per group, **P 0.001).
|
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] |
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