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B The recovery kinetics of heterochromatin in MEFs infected with Flag alone or SKO plus Flag or Gadd45a on day 3. The ratio of MF of heterochromatin is shown at the right panel. More than 19 cells were analyzed for each group. (*p≤0.05)
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(g) Endogenous LC3-II levels in HeLa cells transfected with control or Gsα siRNA for 72 h and treated with or without 400 nM bafilomycin A1 for the last 4 h. Densitometric analysis is relative to actin. Error bars show s.e.m.
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(B) Distribution of GLT-1agold particles in AxT in SI of Ctr KI and Cef KI KI mice. The density of the membrane-associated gold particles (arrowheads) is increased in Cef-treated KI mice (cf Table II).
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A KRAS and TP53 mutation frequencies in MPM from the catalogue of somatic mutations in cancer (COSMIC) stratified by histologic subtype (n = 775 patients).
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(C) The high ratio signal cells were quantified by flow cytometry with dual excitation of mt-mKeima at 633/488 nm for untreated (left panel) and DFP-treated (24 h, right panel) Huh7 cells.
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B. Schematic diagrams of Hog1-WT and its deletion constructs used in this study.
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(A) Quantitative MS-based phospho-proteomic experiments used in this study. Experiments performed in this study compared the phosphoproteome of a wild type and a CDC55 deletion (n = 8) or depletion strain (n = 4), and the effect of hyperosmotic stress (0.5 M NaCl, ten minutes) on global phosphorylation patterns between a wild type strain and a CDC55 (n = 2) or an IGO1/IGO1 deletion (n = 3) strain. Published MS datasets that were integrated (indicated in the upper right) capture the early response to hyperosmotic stress (0.5 M NaCl, five minutes) and the effect of Hog1 inhibition on the early response to hyperosmotic stress (0.5 M NaCl, five minutes) (Romanov et al., 2017, Data ref: Romanov et al., 2017), the effect of a deletion of RTS1 (Hollenstein et al., 2020) and the effect of Cdc28 inhibition (Kanshin et al., 2017) on the global phosphoproteome. All SILAC ratios represent knockout versus wild type, stressed versus unstressed, or inhibited versus not inhibited. After integration of the datasets further analyses were performed using motif & GO enrichment, as well as in vivo biochemical validation.
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A. LC3C specific sensor mCh-AS3_67 is recruited to Salmonella (SFH2) escaping SCV. Salmonella SFH2 strain is carrying a GFP-reporter gene activated by availability of glucose-6-phosphate in the host's cytosol.
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(b) Increased PI(3) KC3 activity by UVRAG. At 48 h posttransfection with Flag-tagged PI(3) KC3 with Beclin1 (lane 1), with Beclin1 + UVRAG (lane 2) or with Beclin1 + UVRAGΔCCD vector (lane 3), WCLs of 293T cells were used for immunoprecipitation with anti-Flag followed by an assay for PI(3) KC3 lipid kinase activity, as described in the Methods. At 16 h posttransfection with p40(phox) PX-EGFP fusion, HCT116.vector and HCT116.UVRAG cells were detected using an inverted fluorescence microscope. p40(phox) PX-EGFP-positive vesicles were quantified as the mean ± s.d. of combined results from three independent experiments. The scale bars represent 10 μm.
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A Cholesteryl ester synthesis, as measured by 3H-oleate incorporation. Note increased CE in ApoE4-treated cells, whereas there was essentially no difference in triglyceride (TAG) production (mean±SE; n=3. with 3 replicates/experiment). †, p=0.06 vs E3.
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(E) Autophagy in neurites at 6 h after SNOC exposure. Arrows indicate autophagosomes engulfing injured mitochondria next to an intact mitochondrion in the left image. The arrowhead in the right panel indicates membrane wrappers characteristic of autophagosomes. White arrowheads in the insets show the double membranes of the autophagosome and those of mitochondria within the autophagosome. Scale bars, 500 nm.
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Images and quantification of colonies formed by GBM cell lines after treatment with different concentrations of EFV (0 - 20 μM). Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0056, ***P < 0.0001; LN18: ***P < 0.0001.
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(F) Representative western blots of fractionated cell extracts of HeLa cells at indicated time after UV irradiation (16J/m2) using histone modification specific antibodies. Loading controls for H3K9 and H3K27 are depicted in figure F
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(E) Mapping of chemical shift perturbation results on the crystal structure of LC3 bound to HsAtg4B. The residues with Δp.p.m. >0.4 are shown in blue, 0.4>Δp.p.m.>0.3 in cyan and 0.3>Δp.p.m. >0.2 in green.
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B Effect of Gli1ZF mutants on the binding affinity to DNA as predicted by in silico alanine scanning. The ∆∆G was calculated along MD trajectories as the difference between the ∆G of each Gli1ZF alanine mutant and Gli1ZF‐WT. Results are shown as ∆∆G values in kcal/mol calculated by means of the MM‐PBSA methods ± SEM.
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Immunoblot of conditioned media collected from HEK293T cells transiently transfected with ERdj3WT or ERdj3H53Q and incubated with an affinity resin consisting of native (N) or guanidinium hydrochloride (GdnHCl)-denatured (D) RNAse A covalently coupled to sepharose resin. Resin was incubated with media for 2 h at ambient temperature, followed by washing and elution in Laemmli's reducing buffer.
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Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Relative CD11b+ MHCII- Gr1hi neutrophil (E) and CD11b+ MHCII- Gr1lo monocyte (F) content in colon (n=13) and tumors (n=16). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.
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(F-Q) Dorsal (F-N) or ventral (O-Q) injection of wt Wip1 abrogates the in vivo expression of mesodermal markers at stage 10.25 as analyzed by in situ hybridization. Embryos are shown in vegetal views with dorsal to the top. Arrowheads denote the absence of expression of mesodermal markers in the injected region of embryos. Control, an embryo injected with LacZ only. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng) and LacZ (100 pg). Scale bar, 150 μm.
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(B) Anti-GFP (upper) and anti-PROPEP3 (middle) immunoblot analysis of PROPEP3-Venus in seedlings submerged in liquid medium and treated with (+) or without (-) 0.5 M Pep2 for the indicated times. The positions of the molecular weight markers are shown on the left. Arrowheads and asterisks indicate PROPEP3-Venus and cross-reactive bands, respectively.
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(A) RT-qPCR analyses of Hlf, Tef, Dbp and Nfil3 expression monitoring changes induced by acute ERS in MPH (3 to 5 independent experiments) (left panel) or mouse liver (5 mice per group) (right panel). The bar graphs show means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05.
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Modulating safeguard with VR. The effectiveness of safeguard can be quantified by the area under the curve (AUC) from density differences between the microbial swarmbot and the surrounding environment. The swarmbot dynamics can be described by a two-compartment model: , and , where the transport of cells between the two compartments is described by the final term in each equation. Modeling predicts that AUC increases with VR, and once VR becomes sufficiently large, AUC approaches infinity, indicating high level of safeguard. As the initial conditions, the parameters are set as: NMSB = 0.1 at t = 0, μ = 1.75, Nm = 1, d = 2, K = 0.01, α = 4, fN = 0.1, VR = variable from 1 to 105. Insets demonstrate the simulated growth dynamics for two representative VR values.
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b, No significant inward current was detected in NMDG+ (Na+-free, Ca2+-free, pH 4.6) solution.
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(B) Hypergeometric testing showed a significant association of nuclear ZEB1 and FOSL1 expression on the protein level as determined by immunohistochemistry on 81 human breast cancer samples. The upper panel shows staining of a serial section of a negative and a positive case. Note that stromal cells (asterisks) are negative for FOSL1, and partially positive for ZEB1 and YAP, and that expression of ZEB1 in tumour cells (arrows) of positive cases is weaker than in stromal cells. Odds ratio (OR): measure of association between categorical variables with OR > 1 indicating a positive association.
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(A) Interaction of external (pExtTseTBg1) and internal (pIntTseTBg1) promoter DNA fragments of TseTBg1 operon with the TsiTBg1 protein. (B) Interaction of pExtTseTBg1 and pIntTseTBg1 promoter DNA fragments of TseTBg1 operon with the HTH domain deleted variant of TseTBg1 protein (TsiTBg1ΔHTH). (C) Interaction of pExtTseTBg2 and pIntTseTBg2 promoter DNA of TseTBg2 operon with the TsiTBg1 protein. (D) Interaction of control (random) DNA with the TsiTBg1 protein.
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] |
C. Quantification of collagen degradation by cells treated with 1 µM 4HT, 4HT + DMSO vehicle, 4HT + 50 µM Blebbistatin or 4HT + 10 µM GM6001 for 16 h. Means ± SEM (n=15), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.
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Percentages of macrophage populations in wild type and Sl/Sl E14.5 brain, skin (dissected from the back), lungs and limb buds, as determined by flow cytometry. Cells were gated as Ter119- CD45+ and further gated into F4/80hi CD11blo or F4/80lo CD11bhi populations. Representative dot plots of skin analysis are shown in Appendix Figure S1A. Data are the mean (±SD) of 4 biological replicates for either genotype, with each replicate consisting of individual or pooled samples (from up to 3 embryos). A total of 8 wild type and 5 Sl/Sl embryos were analyzed over 2 independent experiments.
|
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Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. Timeline of the experimental protocol.
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(C) Gait of chimeric mice. Front and back paws of mice were dipped in red or green paint, and mice walked across a box lined with paper. The npc1−/− mouse displays shorter stride length and a smearing of the footprints as the paws are not as well lifted between steps. Chimeric mice with high amounts of wild-type contribution (C4.8; C4.4 not shown) exhibited a gait indistinguishable from wild-type.
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C) Western blot analysis of AGR2 expression in control and TMED2 overexpressing HEK293T cells upon autophagy inhibition with 20 µM and 10 µM chloroquine treatment. Actin (ACT) served as a loading control.
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HP1b null mutant did not show ectopic vein in the wing and failed to genetically interact with HP1c and Notch. Scale bars, 500 μm.
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(I) Enrichment plots from gene set enrichment analyses (GSEA) performed using LIVER-ID genes as the gene set and transcriptomic changes induced by acute ERS in MPH (upper panel) or mouse liver (lower panel) as the ranked gene list. NES and FDR (as in all subsequent GSEA panels) are the normalized enrichment score and the false discovery rate provided by the GSEA software, respectively.
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G-H. Representative H&E staining and statistical results of metastatic lung nodules from mice injected with the indicated cells via the tail vein for 60 days. (Five sections evaluated per lung). N=8 per group. **P < 0.01, Student's t-test, mean ± SD.
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(M) The graph depicts the percentage of ZIKV (MOI 5, 48 h) infected control and IRGM knockdown HuH7 cells stained with 4G2 antibody analyzed by flow cytometry
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(a) Timeline of tissue collection for analysis of DAPK1 degradation in rats; IHC, immunohistochemistry.
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(S) Total number of eggs (left panel) and number of eggs laid each day (right panel) since hatching by wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes (ordinary one-way ANOVA with Tukey's multiple comparisons test, n=8-12 biological replicates). Data information: mean± SEM points indicate mean± SEM. Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.
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(B) Representative images of Nr2f1 KO Pax6 HET neurospheres obtained from E15.5 neocortices and cultured in vitro. Scale bars: 50µm.
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A) Bar graph of TUNEL-positive cells in the retina of mice treated with 5 mg/kg anti-VEGF-A, anti-ANG-2, and 3, 5, and 10 mg/kg anti-VEGF-A/ANG-2. Error bars show SEM with n = 6 animals per group and * denote all significant changes after one sided ANOVA (**, P = 0.0041) and Tukey's multiple t-test. IgG control is significantly different from anti-VEGF-A/ANG-2 mid (**, P = 0.0077) and -high (**, P = 0.0028). B) IgG control, C) anti-VEGF-A, D) anti-ANG-2, E-G) anti-VEGF-A/ANG-2 low, mid, high: representative micrographs of whole-mount retinae preparations display the reduction of TUNEL-positive photoreceptor cells (green) clustered around focal neovascular lesions in anti-VEGF-A/ANG-2 (E-G) and, to a lesser extent, anti-VEGF-A- (C) and anti-ANG-2 (D)-treated groups compared with IgG-treated controls (B), scale bar 200 μM.
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B, C. Ribbon representation of the PlexA1-4-Sema1b1-2 1:1 (C) and 2:2 (D) complexes. N-glycans are shown in stick representation.
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Statistical analysis of the immunostaining of γ-H2AX (C) in HCT116 cells treated Total nuclear intensities were obtained on >6000 (γ-H2AX) individual cells for each condition from four or two independent biological replicates, respectively. **p<0.01, ****p<0.0001 by Kruskal-Wallis test. A.U. Arbitrary Units.
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c, Log2(H:L) plots for quantified diGly peptides for HCT116PARKIN (experiment 17) or HeLaPARKIN (experiment 57) cells (Supplementary Table 2).
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E Whole-body oxygen consumption rate (VO2) during 24 hr in WT, FADD-D, ob/ob, and FADD-D/ob/ob mice fed a SD. n = 4 for each genotype. Data are expressed as mean ± SEM. *P = 0.0053, **P = 0.0025, ***P = 0.0062, ****P = 0.0011 (one-way ANOVA).
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(D) Immunofluorescent microscopy staining of Fstl1 (green) and α-smooth muscle actin (red). Arrows indicate Fstl1 and α-SMA double positive cells. Scale bar indicates 20μm. Nuclei were stained by DAPI.
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Percentages of axons that initiated regeneration 24 h after laser surgery. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information: statistical significance was determined by Fisher's exact test; *P < 0.05; **P < 0.01; ***P < 0.001; NS; not significant.
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Schematic representation of the MeCP2 protein, with the methyl binding domain (MBD) highlighted in red and the transcriptional repression domain (TRD) in blue. Patients' mutations investigated in this study are indicated.
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A. HeLa cells were transfected with two different siRNAs against NSUN3 (siNSUN3_1, siNSUN3_2), ABH1 (siABH1_1, siABH1_2) or with nontarget (siNT) siRNA and the knockdown efficiency was analysed by quantitative PCR. The relative abundance of the NSUN3 or ABH1 mRNA was normalized to GAPDH levels. Data are presented as mean ± SD.
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B. The truncated receptor encoded by RNA1 is endogenously expressed. HEK293 cells were transfected with a cDNA encoding the human truncated 5HT2C (RNA1). SDS-lysates from these transfected cells and from mousehypothalamus were analyzed by Western blot using an antiserum made against RNA1 (Figure EV1). Mousehypothalamus was exposed 10 times longer. In brain, a band around 75kDa of unknown origin is detected by the antiserum (indicated by a star).
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Double immunolabeling of TRF1 (green) and SYCP3 (red) in squashed spermatocytes from wild-type or Pds5AB cKO mice M-P). The first three columns correspond to z-projections of 15 focal planes throughout the Top, Equator and Bottom regions of the nucleus. The projection of 65 focal planes across the same spermatocyte nucleus is shown at the fourth column (Z-Projection). White arrowheads in (F indicate the presence of TRF1 signals non-attached to the NE. Scale bar, 10 μm.
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E, Representative images of embryoid bodies (EBs) derived from wild-type iPSCs in which the expression of Dnmt3a and Dnmt3b was transiently repressed by siRNAs. Scale bars, 500 μm. Plots show the quantification of the size of EBs and the percentage of EBs with large cavities or beating at different time points during the differentiation process. Data are represented as mean ± s.e.m. (n=3 independent experiments).
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(D) Confocal images of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 together with PTPIP51-HA and RFP-Sec61β and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm.
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A flow chart summary of the actual chain of events triggered by destabilizing mutations in DHFR that eventually lead to bacterial filamentation, as revealed in this study.
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(A) HEK293 cells were transfected with Flag‐tagged beclin 1 and Bcl‐XL with or without haemagglutinin‐tagged DAPK. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies.
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D. HEK293 cells were transfected with C-terminal GFP tagged PTEN and PTENε and subjected to cell fractionation, followed by western blotting with GFP, E-cadherin, and β-tubulin antibodies. C: cytoplasm. M: cell plasma membrane.
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Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in F) pid-5 mutant
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Inner ring (grey): Classical transcription-translation feedback loop or the central circadian clock, involving the transcription activator CLOCK and BMAL1 and the transcription targets/repressors Period (Per) and Cryptochrome (Cry). Outer ring (green/yellow): Model of rhythmic GE driven via AS-decay. AS regulates the formation of productive stable or unproductive instable isoforms, thereby regulating the abundance of SR protein mRNA and protein. An additional regulatory layer is achieved through temperature-mediated control of SR protein activity (active SR proteins are indicated by an asterisks), resulting in rhythmic GE of SR proteins themselves and output target genes. GE in response to continuous (top) or single (bottom) input signal (indicated by arrows) is shown in the center.
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G-H, quantification of vacuoles in E-F. One-way ANOVA was used to analyze statistical differences. Mean with ± SD . Data information: Results are representative of three independent experiments.
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A: Schematic workflow on the collection and processing of surgical specimens collected during a pancreaticoduodenectomy. Samples were provided on ice directly from the operation room and the ME was separated from lamina propria mucosae.
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(D) Comparable variability of RNA and protein levels for genes quantified at both levels.
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Meta-gene plot showing average ISWI enrichment (y-axis = ChIP/Input) in clonal ISWI-3HA parasites at 12 hpi from 1.5 kb upstream of the translation start site ("Start") to 1.5 kb downstream of the translation stop site ("Stop") for the active var gene (PF3D7_1240600, blue) or silent var genes (yellow). One replicate was used for the ISWI ChIP-seq.
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(A) Transactivation assay in HEK293 cells with an integrated 4xSLP-HRE2 E1B 12 TATA Luc reporter and transiently transfected with human WT or W752R AR followed by stimulation with increasing DHT concentrations.
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Knockdown of cyclin F causes a delay in mitotic entry that is dependent on E2F7 and E2F8. Number of Ctrl (left panel) or 7/8KO (right panel) RPE-FUCCI cells with scr or cyclin F RNAi that finished mitosis during live cell imaging is shown. For each condition, 50 cells at G1 were monitored by live cell imaging. Each cell was followed until it successfully progressed through S and G2 phase, finished mitosis and divided into two daughter cells, for a maximum of 24 hours. Log-rank tests were performed to analyze the statistical significance.
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A NSC34cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant PDIs. After 48 h, overexpressed PDI variants was assessed under reducing conditions in an 8% SDS-PAGE. An antibody detecting total PDIA1 (endogenous mousePDIA1 and exogenous humanV5-tagged PDIA1) (first panel). A second antibody detects only humanPDIA1, therefore only V5-tagged PDIA1 appears (second panel). A mouse and human specific antibody was used to detect total ERp57 (third panel). Anti-V5 was used to detect the exogenous PDI variants. Anti-β-actin was used as a loading control.
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Kyte-Doolittle hydrophobicity plots of the complete amino acid sequence of human WT Rab8a and T22N-3IS mutant. The hydrophobicity peak within WT Switch I was abolished in the 3IS mutant (indicated within the red box). The numbers on the horizontal axis denote the corresponding amino acid positions in these proteins.
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Distance of DCVs to the active zone (nm), measured from center of DCV to the middle of the active zone. Histogram of distance of DCVs to the middle of the active zone (nm). Inset: shortest distance of DCVs to the active zone (nm).
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Western blots showing activity changes in both USP7 and USP47 upon treatment with nigericin. MDMs were either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and then treated with nigericin (10 μM) for differing lengths of time, as indicated. Relative, activity-based labelling of USP7 and USP47 was analysed by western blot after incubation with ABPs. Higher panels show lysates incubated with ABPs (labelled), with the active DUB bound to ABP indicated by the suffix '-VME'. Lower panels, without probe labelling, show total USP7 or USP47 levels. (Unlabelled). Western blots are representative of 3 independent MDM donors.
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(C) Panel of WT and KO isolated neurospheres grown from day 1 to day 10. Blue and red lines represent the diameter of WT and KO neurospheres, respectively. Data information Scale bars: 50µm.
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Liver mRNA expression of Growth Arrest and DNA Damage inducible 45 alpha (Gadd45a; A, D, G), beta (Gadd45b; B, E, H) and gamma (Gadd45g; C, F, I) was measured in fed and fasted obese/diabetic monogenic (db/db (n=4/group); A,-C), obese/pre-diabetic polygenic New Zealand Obese (NZO (n=4/group; D-F), and aged C57Bl/6J (22mo; n = 5/group; G-I) as well as corresponding lean, young wildtype (WT) mice (n=4/group). Matched controls for the NZO and aged mice were New Zealand Black (NZB) and 12wk old C57Bl/6J (12wk) mice, respectively.
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(A, B) Poly(I:C) treatment inhibits axonal growth at 3 DIV (A) and dendritic growth at 6 DIV (B) in WT neurons. Neurons were transfected with GFP at 1 or 4 DIV. One day later, neurons were treated with poly(I:C) for 24 h before harvest. (C) Axon and dendrite morphology of Tlr3-/- neurons after poly(I:C) treatment. Data in A,B,C were analyzed by unpaired t-test. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. ** P < 0.001, *** P < 0.0001. Scale bar, 20μm in A and B.
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Schematic model of the dual function of LIF in preventing intestinal inflammation progress.
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B Nucleosome position frequency values for the coding regions of 4013 S. pombe genes were k-means clustered (k = 9) using the abo1Δ data from biorep 1 (low MNase) and displayed with positive values coloured yellow and other values coloured blue (left-hand panel). The cluster order was then used to display the equivalent wild type frequency values in the right-hand panel.
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D, Strain CTD1 harboring a multicopy 3×HA APG9 plasmid was grown in YPD and converted to spheroplasts as described in Materials and Methods. A crude lysate was prepared and treated with or without endo H as indicated overnight at 37°C and analyzed by immunoblots with anti-HA antibodies or anti-CPY serum. Endo H treatment removes the N-linked glycosyl residues of CPY, causing a shift in its electrophoretic mobility. In contrast, Apg9p is not glycosylated and remains unchanged in the presence or absence of endo H treatment.
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(F) ITC profile, representative of at least 2 independent experiments, for biotinylated GCS and GCS Δ3C cytosolic tails with recombinant GRASP55.
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A) Differences in baseline performance and rate of decline on each clinical measure for each participant; the heatmap indicates each participant's standard deviation (SD) from the group mean.
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E. Average expression level of the genes Fas3 (left panel) and zfh1 (right panel) used as markers to assign epithelial and myoblast cells, respectively, in the reference dataset.
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J) Representative respirometry plot of intact cells ± shAMPK and 16 hour BAM15 treatment in C2C12 myotubes cells sequentially injected with oligomycin, FCCP, and a cocktail of rotenone and antimycin a (N=5 per condition)
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D. Projection of confocal sections of inner ear HCs of 5-dpf control (D) and Wrb-deficient pwi mutant fish (wrb pwi/pwi; D'), showing strongly reduced otoferlin immunofluorescence in the mutant HCs. The color look-up table used represents higher pixel intensities with warmer colors.
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(A) Volcano plot of proteins up or downregulated following prolonged palbociclib (1.25μM) treatment in RPE1 cells. The top 10 significantly upregulated and downregulated proteins are shown in blue and red, respectively.
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A. Body weight gain of male C57BL/6 mice that, after 7 weeks in HFD, started receiving treatment with CRT0066101 or water. HFD continued along with treatment. n=8.
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Detection of the number of X-linked Atrx gene foci by DNA-FISH, with or without dox addition for 96 h (N >100 each), performed as described in Figure 1C.
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Three constructs were designed to target GmLMM1 and its close homologous genes. The CRISPR target sequence is shown in orange, and the corresponding protospacer adjacent motif (PAM) site is shown in blue. The box indicates the putative sites shear cut by cas9. Two representative T1 transgenic lines (aged 1 month) are shown for each construct. The editing results were examined by PCR and sequencing; - means deletion of the corresponding nucleotide. The red or green face-labeled letters indicate the mutated or added nucleotides, respectively. (A) Editing of GmLMM1 and Gm13G053800 by construct 1 (C1). The alignment of the sequence and phenotypes of two T1 lines (C1-16-1 and C1-16-5) generated from the same T0 line (C1-16) are shown. (B) Simultaneous edits of GmLMM1, Gm13G053600, and Gm13G053800 by the C2 vector. (C) Editing of GmLMM1 and Gm13G053600 by C9 vector.
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BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors probenecid (1 mM) 20-30 min prior to cell stimulation. To activate the NLRP3 inflammasome, BMDM were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 4 h and stimulated with nigericin (10 μM) for 1 h.
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Acute hippocampal slices of cDKO were pre-incubated for 1 hour either with 10 nM CTα16 or CTα16scr before inducing LTP by TBS (arrowhead). Averaged potentiation levels at LTP induction (t20-25) revealed a significantly enhancement by CTα16 as compared to CTα16scr present over the whole recoding time. Potentiation levels at t75-80 are significantly increased in the presence of CTα16 (**p<0.01). The LTP induction rate is shown as percentage % of mean baseline slope. Data points were averaged over 6 time points. N CTα16 significantly enhances short-term synaptic plasticity at inter-stimulus intervals of 40 (**p<0.01), 80 (*p<0.05), and 160 (**p<0.01) ms compared to CTα16scr.
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(A) Amylose pulldown from benzonase-treated HEK293T cell lysates expressing 2xMBP-BRCA2NT in untreated or irradiated cells (6Gy; +IR). DDX5 and BRCA2NT (MBP) detected by immunoblot. StainFree images of the gels before transfer were used as loading control
|
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Experiments were performed using murine BMDMs. (C) Cell extracts from LPS-stimulated Nfkb1[SSAA] and Nfkb1[SSAA]/Tpl2[D270A] BMDMs were immunoblotted for the indicated antigens.
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(E and F) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, or catalytic-dead PI5P4K2C along with RFP-LC3 for 16 hr (E) or 30 hr (F) were fixed and imaged on a confocal microscope. Bar, 10 μm.(G) Quantification of cells (percentage of total) showing more than ten autophagic vesicles (RFP-LC3 vesicles) in the different conditions from (F) is shown in the graph; n = 200 cells (mean ± SEM). See also Figure S3.
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(C) The same WB membrane containing lysates from R273Hp53-expressing A431cells was incubated with rabbit polyclonal CM-1 antibody and mouse monoclonal 1801 antibody against the N-terminus of p53. Detection using anti-rabbit IRDye 680LT (red) and anti-mouse IRDye 800CW (green) secondary antibodies confirmed that the Δ160p53 band corresponds to a C-terminal isoform of p53.
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The stability of the same samples, as in panel (D), was assessed by NanoDSF. The changes in the 330 nm/350 nm ratio with temperature (left) were used to calculate the first derivative (right).
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A-C) dSTORM images of cells seeded for 6 days with non-neurodegenerative control (non-ND control), FTLD-TDP-A and FTLD-TDP-C seeds extracted from post-mortem brain tissue. Staining shows localization of TDP-43 (HA tag, cyan) along with LaminB1 (yellow, A), nucleopore complexes (nucleopores, yellow, B) and nucleolus (yellow, C). Cells seeded with FTLD-TDP seeds show coaggregation of nuclear membrane proteins upon neoaggregate formation. All images are presented at the same scale, 2 μm as indicated in the non-neurodegenerative control top panels. D) dSTORM images of cells containing aggregates after 6 days of seeding with SarkoSpin fractions of postmortem brain from patients with FTLD-TDP-A or FTLD-TDP-C. Red arrows point to cytoplasmic aggregates. Cells were labelled with TDP-43 (HA-tag, cyan) and phosphorylated TDP-43 pS403/404 (pTDP-43, magenta). All images are presented at the same scale, 2 μm as indicated in the FTLD-TDP-A top panel.
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(A, B, C). RNA-seq transcriptional profiles across the modified locus, as well as immunoblots assessing protein expression of Lon and FtsH are shown for ΔIndLon (A), ΔIndFtsH (B), and ΔIndLon_FtsH (C) grown under inducing or depleting conditions (48h and 72h of depletion for Lon and FtsH, respectively). Symbols +/- indicate inducing or depleting conditions. LC, loading control. WT, wild-type. A schematic representation of the DNA rearrangements in the lon and ftsH locus is also shown for each strain. The ftsH inducible platform inserted by transposon delivery is shown for ΔIndFtsH and ΔIndLon_FtsH strains. The Pxyl/tetO2 inducible promoter is highlighted with a red bent arrow and the terminator sequence used to isolate the promoter is represented by a hairpin structure. The tetR repressor gene and the resistance markers cat and tetM are indicated in purple, blue and red, respectively.
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F Control HeLa cells (transfected with a scrambled shRNA) or cells depleted of DDX6 were transfected with a mixture of three plasmids: the R-Luc-8xlet-7-poly(A) or the corresponding reporter carrying mutations in let-7-binding sites (R-Luc-Mut, Mut), a plasmid expressing F-Luc as a transfection control, and a plasmid expressing shRNA-resistant versions of GFP-DDX6 (wild-type or the indicated mutants) or MBP. For each condition, Renilla luciferase activity was measured, normalized to that of the F-Luc transfection control and set at 100% in cells expressing R-Luc-Mut. The left panel shows the normalized Renilla luciferase activities in control cells (i.e., cells treated with scrambled shRNA and expressing MBP). The right panel shows relative fold derepression for each condition for the R-Luc-8xlet-7-poly(A) after normalization. Mean values ± standard deviations from 4 independent experiments are shown.
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D Clonogenic assays in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and irradiated at the indicated X-Ray doses, then plated at low density. After 9 days, colonies were stained with crystal violet and counted (mean ± SEM; 3 independent cell lines per genotype).
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(C) Representative example of immunofluorescence staining of N protein (green) after SARS-CoV-2 infection of Calu-3 at MOI 0.4 TCID50VERO/cell, at time points shown. Nuclei (DAPI, blue), cell mask (red). Scale bar represents 50µM.
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(B). Structures of the three compounds (DB1246, DB1247, DB1273), highlighted by green circles in A, that show preferential binding to RNA (G4C2)4 G-Qs and were further characterized.
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(A) Overlap of phosphoproteins that were "up-regulated" by nitrate at 5 min (dark yellow) and 20 min (light yellow)
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A, B Oil Red O staining of livers from WT and KO males after 25 weeks on NC or HFD, with quantification of relative stain intensity (n=3, 3 images each). Scale bar = 100 μm. Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).
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. D. Nuage specific markers (Aub, Ago3 and Mael) are shown in stage 3 wild-type and germline mutant clones for Rpp3018.2. Scale bar, 10µm.
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(E) IDH1 K224 deacetylation led to degradation of HIF1α but upregulation of hydroxy-HIF1α. In HCT116 stable cells with IDH1 knockout and re-expressing the indicated proteins, HIF1α and hydroxy-HIF1α was measured by Western blot assay.
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(E) Dissociation of tRNAfMet(Flu) from 80S 2C deacylated tRNA incubated with eEF2 and eEF3 (green) or eEF2 (red) for 15 min before adding with ternary complexes. Each trace is an average of 5-7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction.
|
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(B) Representative images of PKH-67 (green) and DAPI (blue) staining in MuSCs freshly isolated from muscles previously injected with PKH67-labeled EVs. Scale bar = 25 μm. Data information: Nuclei were counterstained with DAPI (blue).
|
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(D) Immmunofluorescence images of endogenous ACTN4 in non-dividing and post-mitotic NIH3T3 and RPE-1 cells in as indicated. Shown are maximum intensity projections. Cells were fixed in 4% PFA and stained for ACTN4 and nuclei (DAPI) as indicated. Scale bars 10 μm.
|
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(F) Representative images of H&E, CK19 and α-SMA IHC staining performed on lung sections. Arrow identified lung metastasis. (G) Quantification of lung metastasis size and number based on CK19 positive staining using NanoZoomer software. Values are means ± SEM of 6 mice per group.
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