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(G) HeLa CCL2 cells were transfected with either wild type Flag−β-catenin (+WT), Flag-β-catenin S60A mutant (+S60A), or Flag-β-catenin S60D mutant (+S60D), then treated with 200 ng/mL nocodazole for 18 h (metaphase) and released by incubation in fresh medium for 2 h (telophase). Cell lysates were used for pull-down analysis with rhotekin-RBD beads, and the pulled down beads were separated by 12 % SDS-PAGE followed by immunoblot analysis with the indicated antibodies. The membrane was then stained with Coomassie (CBB) to determine the amount of RBD precipitated.
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A Kaplan-Meier plot depicting metastasis onset after tumour removal in mice injected orthotopically with HR1 gCtrl (n = 13) or HR1 g4Nfib (n = 14), two-tailed log-rank test.
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(H-J) RNA isolated from control and IRGM knockdown HT-29 cells and subjected to qRT-PCR with indicated genes of (H) Immunoproteasome complex (I) TAP complex (J) human leukocyte antigen (HLA) system.
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(d) Histogram of single-cell TDP43(A315T)-EGFP levels. Asterisks in b and d denote median values for cells exposed to vehicle (gray), FPZ (green), MTM (blue) or NCP (purple). For FPZ, MTM and NCP versus DMSO in a and d, P 1 × 10−3 by two-sided Kolmogorov-Smirnov test.
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F-G, drug tolerant rate of (F) Du145/Du145TXR (20 nM taxol/20 nM taxol plus 50 μM verapamil) or (G) MCF-7/MCF-7ADR (200 nM adriamycin/200 nM adriamycin plus 50 μM verapamil) cells treated with drugs on the indicated days. Drug tolerant rate defined by comparing cell viability followed treatment, RMCs VS. MCs or CCs VS. PeCs, > 1 representing drug tolerant. Induction Time indicates the time of entering DTP state; Sustaining Time indicates the time of maintaining DTP state. Blue colors represent control groups; Red colors represent MDR cancer cells. Mean with ± SD.
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Ileum was isolated from non-stimulated GRfl/fl (n=4) and GRVillinKO mice (n=4) and gene expression was measured via qPCR. Expression levels are shown as fold induction with GRfl/fl expression values set at 1. P-values were calculated using a student's t-test.
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qRT-PCR and Western blot analyses of ACTA2/αSMA expression using total RNA/lysates of fibroblasts treated with activin A or TGF-β1 for 6 h. N=3. The membrane was re-probed with GAPDH and mDia2 antibodies.
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(e) Western blot analysis of GFP and alpha-tubulin (control) in cells transfected with GFP-ORF1p and treated with control or Bafilomycin (20 h, 400 nM).
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a, Alteration of both LC3 conversion and translocation in Ambra1gt/gt embryos. Left panel: immunoblot analysis of LC3 in E14.5embryos. LC3-I to LC3-II conversion is reduced in mutant extracts (LC3-II/LC3-I ratio, as measured by densitometry, from left to right: 5%, 9.8%, 2.5%, 4.6%). B, body; H, head. Centre and right panels: sections from E10.5Ambra1+/+;GFP-LC3 and Ambra1gt/gt;GFP-LC3neuroepithelia were compared to evaluate GFP-LC3 subcellular distribution. Clusters of GFP-LC3 punctate structures (arrows) are indicated in wild-type sections. Images were optimized by deconvolution. Scale bar, 10 µm.
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(I-J) VEP recording images (I) and N2-P2 wave amplitudes (J, n = 5 mice) show the difference in the VEP N2-P2 wave amplitude between wild-type and Enkd1 knockout mice.
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(A) Average expression of Dppa2 and Dppa4 in a single-cell RNA-seq analysis of mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population.
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D,E. The expression of primary miR-23a~27a~24-2 transcript during EB formation (D) and RA induced differentiation (E). Error bars indicate s.d. (n=3).
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E) quantification of lung lesion burden from H&E-stained sections Data information: Data are means + SD of the indicated number of mice from 1 of n=3 independent experiments and each symbol represents data from 1 mouse. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001. Scale bar, 200 μm.
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Brown preadipocytes were knocked down Raptor following standard induction to mature adipocytes with or without asparagine supplementation. Protein expression analysis was performed on day 6.
|
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D. Colon organoid cultures in presence of ISEMFs with or without Noggin (Nog). Scale bar = 50 μm
|
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Diploid and tetraploid cells containing a fluorescently labeled version of the transcription factor Cap1 (Cap1-mNeonGreen) were grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm. Quantitation of the percentage of diploid and tetraploid cells with activated Cap1 on YPD or PRE-SPO medium. "n.d." indicates that no cells with activated Cap1 were detected.
|
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C, immunoblot analysis of NPC1L1 and NRF2 in Du145TXR or MCF-7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents for 24 hours. Data information: Results are representative of three independent experiments.
|
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L. The levels for IP3R3 and calreticulin were decreased in the MAM fractions of Sig1R−/−mouse brains. MAM fractions and whole tissue lysates of Sig1R+/+ or Sig1R−/−mouse brains were immunoblotted. Representative blots from three independent experiments are shown.
|
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] |
(D) RAW264.7 cells were infected with HSV-1 (KOS), PRV (vBecker3) and the corresponding mutants lacking ICP27 (UL54 in the case of PRV). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.
|
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(f) NRK cells were starved for 4 h, autolysosomes were purified by density fractionation and the quality of purified autolysosomes was monitored by TEM (left panel). Right panel, purified autolysosomes stained with antibodies against PI(4) P and PtdIns(4,5) P2. Scale bar, 500 nm.
|
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(E) Dynamics of spindle elongation. The spindle was visualized with EGFP-Map4 The length/width ratio of the spindle was measured after 3D reconstruction (n=13 oocytes from 3 independent experiments).
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(A) Levels of LC3 and p62 in brain and spinal cord extracts from Epg5+/− and Epg5−/−mice. SC, spinal cord.
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(B) In vitro ADAM17 sheddase activity is blocked by 4D9-effectorless mAb and 4D9 Fab fragment but not an isotype control. Fluorescence polarization of FAM conjugated TREM2 stalk peptide was detected in the presence or absence of ADAM17 and 4D9 mAb, 4D9 Fab and isotype control. Data represent the mean ± SEM (n=6). One-way ANOVA, Tukey's post hoc test; p (4D9 Fab vs 4D9 mAb) = 0.8855; p (4D9 Fab vs uncleaved) < 0.0001; p (4D9 mAb vs uncleaved) < 0.0001; n.s., not significant.
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E Ten days after intracranial transplantation of GB2 cells (1.0 × 104 cells), AK7 was intraperitoneally administrated for 4 weeks (15mg/kg, twice/week). After eight weeks, mice (4 or 5 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA was quantified by qRT-PCR.
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(A) 786-0 cells were transfected with individual siRNAs that targeted 335 E3 ligases (three siRNAs per gene; indicated as 'siGene' on the x axis) for 48 hours and treated with aa starvation for 12 hours in duplicates. Next, the intensity of GDH1-GFP in each well was recorded using Opera Phenix (Perkin Elmer). The data are presented as a volcano plot. The siRNAs with P < 0.0001 (two-tailed Student's t-test) and a GFP intensity ratio (normalized to the non-targeting siRNA) >16 were considered to be effective siRNAs (red). E3 ligases targeted by more than two effective siRNAs were selected as candidates. siE3s, E3 ligase siRNAs; siNC, non-targeting control siRNA.
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E. Increased amounts of ICAT plasmids were transfected into 293T cells. After 36 hr, cells were treated with 50 ng/ml Wnt-3a for 6 hr. Cell lysates were then subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.
|
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Strong nuclear p53 signal in immunostainings of HGSOC organoids is indicative of a gain-of-function TP53 mutation. Scale bars: 100 µm (Tissue) and 50 µm (Organoid).
|
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C. The level of LRP6 was not reduced during nutrient starvation in mouse brain. Mice were starved and brain tissues were analyzed by immunoblotting.
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D) Average I/V relationships (left) and steady-state activation curves with superimposed Boltzmann's fittings (right) under baseline conditions (black) and in the presence of 5 µM LUF7346 (red). * = p<0.05. N: 19.
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Instantaneous growth rates were extracted from the Aci2 monoculture data A variation in Aci2's lag time is clearly visible in this representation. Data Information: Data were collected at 600nm.
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(I) The levels of IL-18 (I) in the serum was measured using ELISA (n=8 mice per group).
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(C) Lung cancer A549 cells, human normal bronchial epithelial cells (HBEpc), and HBEpc-differentiated cells (airway organoids) were harvested for immunoblotting with the indicated antibodies.
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(C) Atg8WT-HA, Atg8L50A-HA or Atg8Y49A-HA was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and cell extracts were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblot analysis using anti‐HA antibodies (lower panel; asterisk represents nonspecific band).
|
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G. Measured MSD (⟨∆r2 (t)⟩) as a function of delay time t for the mobile lysosome trajectories taken at F.
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G-H: Flow cytometric analysis of TOMM20 expression level (G, n=4, technical replicates per clone) and specific TFAM level (total TFAM/TOMM20) (H, n=4, technical replicates per clone).
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(A) MDMs were infected for 18 h with HSV-1 (KOS), MOI 1. Cytosolic viral proteins were detected by mass spectrometry using iTRAQ-labeling. Identified viral proteins are shown.
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Tfeb expression in the vasculature (mice n=10). (A) Representative images of retinal vessels (p5) (A) (scale bar: 50 µm) of Tfeb-EGFP mice stained with anti-iB4 (A) and anti-GFP
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(E) Semi-quantitative RT-PCR analysis of RPE functional markers in cultured porcine RPE cells. GAPDH was used to confirm the cDNA quality of all the samples
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E FST expression and FLT3/ITD signaling were detected by Western blotting in Ba/F3-parental (P in short) and Ba/F3-FLT3/ITD (ITD in short) cells.
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(c,d) WT and Hace1−/− MEF as well as Hace1−/− MEF stably transfected with WT HACE1 and C876S-mutant HACE1were subjected to protein aggregates clearance assay by pulsing with puromycin for 4 h to induce protein aggregate formation followed by an 8-h chase to monitor for aggregate clearance. Cells were fixed and stained for ubiquitinated proteins to visualize aggregates. Vehicle (PBS)-treated MEFs were used as control. (c) Representative confocal micrographs of images used for quantification of the efficiency of aggregates clearance in d (percentage of cells that cleared all aggregates by 8 h after puromycin removal). At least 10 images from each group were used for the calculation, error bars represent s.e.m., P0.001 (one-way analysis of variance). Scale bars, 10 μm.
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A 3-D reconstructions of propidium iodide (PI)-stained cell walls (red) and BCECF-stained vacuoles (green) of epidermal atrichoblasts in the early and late meristem and in the early and late elongation zone. Scale bars: 5 µm.
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(A) Representative immunofluorescence images of KT alignment efficiency in mitotic S2 cells expressing PoloWT-EGFP, PoloT182D-EGFP or lacking expression of any Polo transgene. Cells were treated with MG132 prior to fixation to prevent cells from exiting mitosis and increase the number of pre-anaphase figures. Insets display magnifications of the outlined regions, which highlight both low-tension misaligned (inset 1 and 2) and high-tension aligned KTs (inset 3). (B) Graph represents the percentage of cells in each indicated mitotic state, as shown in (A) (n≥ 532 cells for each condition, n=2 independent experiments). Data information: Data are shown as mean ± SD. Scale bar: 5 μm.
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D. Analysis of relative VEGFA protein release in human control and SIGLEC11/16 knockout THP1-macrophages. Released protein levels were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and polySia avDP20 (0.15 µM and 1.5 µM) in the human macrophages. Data show mean +/- SEM. * p < 0.05, *** p < 0.001, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=9 and p<0.0001, PolySia avDP20 1.5 µM n=6 and p=0.009, LPS n=9, LPS/PolySia avDP20 0.15µM n=6 and p=0.043, LPS/PolySia avDP20 1.5µM n=7 and p=0.0001. Siglec11/16 KO: no treatment n=9 and p=0.349, PolySia avDP20 1.5 µM n= 6 and p=0.249, LPS n=7, LPS/PolySia avDP20 0.15µM n=6 and p=1.0, LPS/PolySia avDP20 1.5µM n=7 and p=1.0.
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(A) (B) CPT sensitivity assayed by 10-fold serial dilutions of different mutant combinations on YPAD plates. Four biological replicates have been performed.
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N (Left) TUNEL assay of the same liver as in (M). Scale bar = 40 μm; Zoom box, scale bar = 5 μm. (Right) Quantification of apoptotic cells. 10 random fields were counted for each TUNEL-stained tissue sample. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-tailed Student's t-test).
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a) Heat shock proteins, HSP90AA1 and HSP90AB1, were destabilised during SARS-CoV-2 infection of Caco-2 cells (circular plots, left side (n=3); see 1A for circular plot legend). Significant protein changes (abundance and thermal stability) are represented as circle plots for individual proteins, as previously described (Becher et al, 2018). Inner circle corresponds to protein abundance and the outer circle corresponds to stability changes (n=3). Asterisk denotes significant regulation (see methods).
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(C) hierarchical clustering of the long noncoding transcriptome of undifferentiated and differentiated adipocyte samples, n=3 (biological replicates), euclidean distance.
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(A) Genotyping of chimeric mice. Upper gel: PCR to amplify a fragment of the npc1 gene from npc1−/−↔GFP, npc1+/−↔GFP, and npc1+/−mice results in npc1− (1,067 bp) and npc1+ (947 bp) bands. Lower gel: The three genotypes can be distinguished by ApaLI digestion of the PCR products. The npc1− allele gives rise to 937- and 130-bp bands. The wild-type allele varies depending on the source: the BALB/c allele is digested to 477-, 340-, and 130-bp bands while the allele from the GFP mouse is digested to 817- and 130-bp bands. Note that the 477- and 340-bp bands are absent from the homozygous mutant chimera.
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(B) HeLa-GFP-Parkin cells were transfected with siNDP52 or scrambled siRNA. Seventy-two hours after transfection, cells were incubated with 1 µM valinomycin for the indicated times. Western blotting was performed using the indicated antibodies. Data are representative of three independent experiments
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Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. D Quantification of mitochondrial mass using MitoTracker green, indicated by MFI FITC in human CD4+/CD8+ T cells +/- CD3/CD28 stimulation, n=5/6/6/6 biological replicates. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.
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A Scheme of the lentiviral vector with inducible FlagOmomyc (top) and immunoblotting (bottom) of FlagOmomyc and actin loading control upon Dox treatment of BT168FOcells for 0-48 hours.
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D. Ty1 insertion profile upstream of tDNAs. Total genomic DNA extracted in (B) was prepared for Ty1 de novo integration event sequencing. Ty1 insertions are computed in a 1kb window upstream of all the 275 nuclear tDNAs (position 0 in the graph). Each position is divided by the number of insertions at this position (weight). The Smoothing curves indicate the general trend. Nucleosome center positions for the three first nucleosomes upstream of all tDNAs are from (Brogaard et al, 2012).
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(S-ZA) C2C12 cells were transfected for 24 h with Jumpy CS and Cherry‐LC3 (LC3), fixed and immunostained with anti‐lamp1. Jumpy CS (green), LC3 (red) and Lamp1 (white in T, X or blue in V, ZA). Boxed areas (S-V) are shown at higher magnification in the corresponding panel below (W-ZA). Yellow arrows indicate colocalization between Jumpy CS and LC3 but not Lamp1. Scale bars, 2 μm.
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(F-G) EM analysis of cho2opi3 cells that after preculture in SD C+, were transferred to OD600 0.05 and subsequently cultured to mid-log phase in SD C+ (F) or SD C- (G) both containing 0.05 μg/mL SorA. CW, cell wall; ER, endoplasmic reticulum; M, mitochondria; N, nucleus; V, vacuole; *, lipid droplet. Scale bars correspond to 500 nm.
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(b) HEK293 cells transfected with Beclin-1 ATG14L were grown in the presence or absence of amino acids. Lysates were immunoblotted with the indicated antibodies (left panel) and quantified by densitometry (right panel). Data represent mean+s.d. of three unique experiments.
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Direct binding of compounds to Munc18-1. Bead-bound fluorescence of compounds 9, 10 and 13 was quantified upon incubation of compounds with bead-immobilized GST, or GST-tagged WT, R406H or G544D. Data are means ± SEM (**p < 0.01, ***p < 0.001, by Kruskal-Wallis test and Dunn's multiple comparison test, or by one-way ANOVA followed by Dunnett's post-hoc test; n = 9 independent experiments; exact p values are shown in Appendix Table S1).
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(D) Untreated Huh7 cells or those treated with DFP (10 μM), DFX (10 μM) or DFO (10 μM) were quantified for mitophagy using mt-mKeima transfection and FACS analysis (n=4, biological replicates). A high signal (633/488) area/ cell area indicates the proportion of cells undergoing mitophagy. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. **: P<0.01.
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E Co-immunoprecipitation of USP34 with ectopically expressed FLAG-tagged RUNX2 in 293T cells
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C Anti-FLAG antibodies are not able to dissociate HSE-DNA-bound Hsf1. Red lines in the cartoon indicate the Hsc70 binding site; blue lines and arrow head indicate the inserted FLAG epitope DYKDDDDK. Hsf1_i201/211/221, FLAG-epitope inserted after residue 201, 211 or 221. Anti-FLAG antibodies were split in halfmers by incubation with 2 mM DTT
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Immunofluorescence analysis of LAMP1 with and without IFNγ in RH, RH+GRA15WT. All the experiments were done 3 times with each of the strains.
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(B). Comparison of residues of PTP-MEG2 interacting with NSF and MUNC18-1. Amino acid residues of NSF and MUNC18-1 are coloured as follows: green, residues interacting with both NSF and MUNC18-1; red, residues specifically contributing to NSF recognition; blue, residues selectively contributing to MUNC18-1 interaction.
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B, phase contrast images of the morphological changes in MDR cells, residual MDR cells and regrown cells for Du145TXR or MCF-7ADR. Red circles mark magnified areas (bottom). Scale bars, 50 μm (low magnification images).
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(C) Alcian Blue and Nuclear Fast Red staining of WT (left) and Hnf4g KO (right) small intestinal organoids. Cells are visualized using Nuclear Fast Red. Intra- and extracellular mucus that is produced in goblet cells stain positive for Alcian Blue
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Ai854517 and Polg transcripts colocalize in adult mouse brain; in situ hybridization. HC, hippocampus; cerebellar molecular layer (M), Purkinje cell layer (PC), granular cell layer (GC). Scale bars: hippocampus 760 µm, cerebellum 300 µm.
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K CHH methylation levels of the RDR6‐RdDM targets TAS3a and Athila6A TSS in whole‐inflorescence‐tissue samples compared to dissected young buds corresponding to floral stages 6-8.
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(e) Immunoblots for Atg5, Atg7 and p62 confirm protein depletion and autophagy inhibition in d,f.
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The biological activity of LV-mediated IL-1Ra was assessed in vitro. RAW264.7 macrophages were transduced with LV.IL1RN or LV.GFP vectors at a MOI of 10. After 72 hours cells were stimulated with recombinant hIL-1β and the expression of TNF-α assessed after a further 48 hours. (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by two-way ANOVA with Bonferroni's post-test; *P<0.05, **P<0.01, ***P<0.001. Symbols above bars are versus NTC within a treatment group.
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F. Immunoblot analysis of the phosphorylated and inactive forms of IRF3, IKBα, TBK1, RIG-I, MAVS, HDAC6, and β-actin at the indicated times (0, 2, 4, 8, and 16 h) in HDAC6+/+ and HDAC6-/- BMDMs. BMDMs were stimulated with PR8-GFP (MOI = 3).
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(C-E) Gene set enrichment analysis of gene expression changes between WT Treg cells vs CD4+ naïve T (upper panels) and WT Treg cells vs Tet2/3 DKO Treg cells (lower panels) for the hallmark gene sets IL2/STAT5 signaling (C), E2F Target genes (D) and Treg signature gene set that are upregulated in Treg cells compared to conventional T cells (GSE7460) (E). The green line represents the enrichment score with enrichment in WT Treg cells shown to the left and enrichment in CD4+ naïve T (upper panels) or Tet2/3 DKO Treg cells (lower panels) shown to the right. Overlap of genes in the given gene set is shown below and denoted by black ticks.
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Unsupervised identification of differentially abundant high-dimensional cell populations between clinical groups was performed using CITRUS (FDR<5%). between P. falciparum symptomatic infected individuals and asymptomatic participants or healthy immune controls. A-E. tSNE plots of clinical groups and viSNE plots depicting expression of selected surface markers, relative abundance and cellular phenotype of differentially abundant clusters identified among CXCR3+CCR6- memory CD4+ T cells (A), CXCR3-CCR6- memory CD4+ T cells (B), circulating memory TFH cells (C), classical MBCs (D), and atypical MBCs (E). The tSNE plots in the top or each panel display cell density and represent pooled data for each group as calculated in the clustering analysis while the viSNE plots on each top panel depict relevant marker expression on tSNE overlays. The lower panels from left to right show differentially abundant populations identified in purple on a tSNE overlay, whiskers showing the range (minimum to maximum), with lines representing the median of 16 (symptomatic), 24 (asymptomatic) and 24 (healthy immune controls) biological replicates, while the pink histograms illustrate marker expression in identified differentially abundant populations, relative to background expression, shown in lilac.
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Mpc2C3 forms a carrier complex with Mpc1, as analyzed by BN‐PAGE. When co‐expressed with Mpc1, Mpc2C3 shows the same complex pattern as Mpc3, including the 300K complex.
|
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Quantification of fst expression by WISH (N) after FLT3/ITD overexpression in zebrafish embryos at 6 hpf.
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(A) Mitochondrial respiration measured in adherent MEFs of the indicated genotypes using Seahorse FluxAnalyzer. Oxygen consumption rate (OCR) normalized to protein concentration. Following basal respiration, cells were treated sequentially with 1μM Oligomycin (Omy), 2μM CCCP, Antimycin A 1μM + 1μM Rotenone. Bar graphs of (A) representing basal (B) and maximum (C) respiration. Data represent mean ± SEM of 7-12 independent OCR measurements, One-way ANOVA; *p < 0.05, ** p < 0.01, ***p < 0.001, ns; not significant. Mitochondrial membrane potential measured by fluorescence microscopy in WT, Opa1Crispr, Opa1Crispr + pLenti-Opa1, Opa1CrisprPgs1Crispr, Opa1CrisprPgs1Crispr + pLenti-Pgs1, Pgs1Crispr and Pgs1Crispr MEFs + pLenti-Pgs1. Membrane potential is represented as the ratio between TMRE/mitoYFP. WT MEFs treated with 20μM CCCP serve as a negative control for TMRE. Data represent mean ± SD of three independent experiments, number of analyzed cells indicated in inset, One-way ANOVA; **p < 0.01, ***p < 0.001, ****p < 0.0001, ns; not significant.
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G, Pie chart showing nonCM composition (erythrocytes and thrombocytes excluded).
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B The distribution of positioned nucleosomes (derived 138-161 bp size class) and sub-nucleosome fragments (derived 112-137 bp size class) for pl-iPSC and iPSC-derived NPC (NPC). Actual calculated numbers are presented above each column.
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Scheme of the migration pattern of the bubble‐, single Y‐, double Y‐ and X‐shaped replication intermediates in 2D gel electrophoresis.
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G. QPCR analysis of the proliferation markers Ki67 and c-myc in ileal lysates from late disease stage Casp8WT and Casp8ECko mice (n= 4 WT, 3-4ECko, two-way ANOVA with Sidak method). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.
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(b) UVRAG bridges the interaction between Beclin-1 and ULK1. HEK293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated. Lysates were immunoprecipitated with anti-HA(Beclin-1) antibody and blotted with the indicated antibodies. A representative experiment of three repeats is shown. Uncropped images of blots are shown in Supplementary Fig. S4.
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Western blot detection of indicated proteins in lysates of mBMDMs from WT and Cgas KO mice stimulated with ecGAMP (5 μg/ml), icGAMP (0.1 μg/ml) or transfected with ISD. Data are representative of 3 independent experiments.
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(b) Representative images of MEFs infected with the GFP-LC3 adenovirus and stained with anti-TOM20 (red) to visualize mitochondria 10 h after mitophagy induction; original magnification, × 400 and × 1,000; scale bar, 100 μm and scale bar, 15 μm.
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(D) Polysome profiling analysis performed in BY4741 wild-type and nat4Δ strains. The plots are representative of two independent experiments.
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(G) Routine (R) oxygen flux, leak (L), OXPHOS (P) and ETC (E) capacity in permeabilized mixed gastrocnemius fibers (N=6 per group). PM: pyruvate and malate, ADP: adenosine diphosphate, GS: glutamate and succinate, Tm: tetramethyl-p-phenylenediamine, PalM: palmitoylcarnitine and malate, Oct: octanoylcarnitine, and Dur: duroquinol.
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H. Glutamate treatment (30 μM glutamate and 1 μM glycine for 10 min) was also associated with a significant loss of mitochondrial density in axons at 6 h later, which was reversed by Miro1-EF but not by Miro1 WT (n = 60-90 axons from 6-9 dishes; **P < 0.01, ***P < 0.001 and ****P < 0.0001, Kruskal-Wallis test)
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E-F, Flow cytometric analysis of PD-L1 (E) and PD-L2 (F) on TAMs in MC38 tumours receiving treatment as indicated above. Data presented as mean SEM and analysed by Mann-Witney (n=5 mice/group)
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A. DNI efficiencies of ΔPrPs are inversely correlated to deletion size. Representative immunoblot showing PK-resistant PrPSc moiety of (3F4) MoPrP co-expressed with indicated ΔPrPs in 22L-ScN2a cells. Quantitative analysis is shown below as scheme. Relative PrPSc levels were calculated as proportion of PK-resistant (3F4) MoPrP co-transfected with each ΔPrP to that of co-transfected with empty vector control in the same experiment. Data from 4 (for Δ159-165, Δ159-167 and Δ159-167(169) only 3) independent experiments were statistically analyzed for mean and standard deviation (error bars).
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G. Representative blue native immunoblots (BN) of Skm mitochondrial membrane proteins from 2-month-old mice treated with edaravone for 30 days (wt + edaravone, n= 3; LowOXPHOS + edaravone, n= 3). The migration of the respiratory complexes/supercomplexes CI-CIV and hATPIF1 is indicated. VDAC is shown as a loading control.
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D, Flow cytometry analysis of MET in BT308NS weekly treated with IR (2 Gy) for 6 weeks. Dotted lines: threshold to define the percentage of MET-expressing cells. Ctrl: non-irradiated cells.
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C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. (A) Chemical structures of heme (HO-1 substrate) and CoPP (HO-1 in vivo inducer).
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B. Cycloheximide chase analysis on N2a cells expressing Sig1R-FLAG. Quantitative data of immunoblotting for the levels of Sig1R-FLAGprotein and its variants during the cyclohexamide chase were plotted as mean ± SEM of three independent experiments (upper panel). Representative immunoblots for the levels of Sig1R-FLAGproteins were shown (lower panel). **: p < 0.01, *: p = 0.0216 in E102Q vs. wild-type (WT); ##: p < 0.01, #: p < 0.05 in L95fs vs. WT. A one-way ANOVA with subsequent post hoc Tukey's test.
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(J) Results of PACE in MZznf598 zebrafish embryos. Black circles show the relative stability of the reporter mRNAs (averages of two biological replicates) in MZznf598 embryos. Gray circles show the relative stability of reporter mRNAs in wild-type embryos The stability of a codon-tag reporter with a UGA stop codon was set to one. Error bars represent maximum and minimum data points. The relative effect of each codon on mRNA stability in wild-type embryos is shown as a color gradient from red (destabilizing) to blue (stabilizing).
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(A) The interaction between EPCR1 and ARID2 or ARID3. Arabidopsis plants carrying EPCR1-Flag and ARID2-Myc or ARID3-Myc transgenes were used for co-IP.
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a, Glycaemia drop in RagA+/+, RagAGTP/+ and RagAGTP/GTP and recovery in fasted RagA+/+ and RagAGTP/+ but not in RagAGTP/GTP neonates (+/+: n = 5, 18, 4, 5, 9, 8; G/+: n = 10, 26, 10, 13, 26, 21; G/G: n = 7, 20, 9, 10, 16, 11, for 0, 1, 2, 3, 6 and 10 h, respectively; mean ± s.e.m.).
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(E,F) Progeny virus released from untreated Vero-E6 cells or Vero-E6 cells treated with clinical doses of hrsACE2 (5 and 10µg/ml) and remdesivir (Remd. 4µM). Progeny was determined (E) 15 hours and (F) 48 hours post-infection (hpi).
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D. Protrusions (Ps) and cell bodies (CB) of cells induced to migrate towards LPA were isolated and analyzed to detect the indicated proteins (by Western blot; left panels) or RNAs (by RT-ddPCR; right panel). Ps/CB enrichment ratios from 2 independent experiments are shown. Bars: mean ± s.e.m..The enrichment of pY397-FAK serves to verify the enrichment of protrusions containing newly formed adhesions in the Ps fraction.
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(H) Death rates in TNF-treated indicated cell lines including isogenic IκBα/IκBε-knockout population (mean of three independent experiments ± standard deviation).
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Confocal images of tPH-CynA-KikGR expressing wild-type Dictyostelium AX3 cells before (left) and after (right) LY2924002 treatment.
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C) Representative images of a Fis1-Drp1 PLA in DKO MEFs expressing either GFP, GFPv1 (GFP-tagged variant 1 of Miro1) or GFPv4 (GFP-tagged variant 4 of Miro1) n=30 cells per condition over three independent experiments). Scale bar is 10 μm. Red dots indicate interaction of Fis1 and Drp1. Blue is DAPI. D) Quantification of PLA fluorescent dots per cells between GFP, GFPv1 or GFPv4 transfected DKO MEFs (n=30 cells over three independent experiments). Data information: In D) a one-way ANOVA with a Newman-Keuls post-hoc test was used to calculate statistical significance. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM.
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] |
(C) Tumor volume in SCIDmice treated or not with LPS n (number of animals per group)=5.
|
[
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B. Cells were EdU-labeled for 24 h (day 10-11) prior to analysis by flow cytometry to quantify proliferating cells. Percentage of EdU+ cells is indicated (n=4; mean ±SD; *p ≤ 0.05, Mann-Whitney U-test).
|
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GSEA analysis based on 145 excluded genes indicates processes that cannot be analyzed with the pipeline.
|
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