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(H) H&E and Oil Red O staining of liver sections from wild-type or JFKTG mice fed on ND or HFD. Scale bar, 100 μm.
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C Western blot analysis of USP33 levels in the spleen tissues of mice fed an NSD or HSD for 7 days or 30 days as indicated. Data information: Data are representative of at least two biological replicates
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(C) HeLa cells stably expressing silent GFP‐LC3B or GFP‐GATE‐16 were transfected with their siRNA pools using DharmaFect reagent. After 72‐h interval, the cells were incubated for 2 h in EBSS medium and subjected to immunostaining with anti‐Atg16 and anti‐p62 antibodies followed by fixation. The cells were analysed by confocal microscopy. Arrows represent cells, which do not express GFP proteins whereas arrowheads represent cells, which express GFP‐proteins. Quantification of Atg16‐labelled puncta structures from three independent experiments is presented at the right panel. Scale bar: 20 μm.
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IL-1β concentration (pg/ml) in supernatants from macrophages of SC naïve (blue; n = 7) and COVID-19 patients (red; n = 11). Macrophages were pre-treated with DMSO (SC-naïve n = 7; COVID-19 n = 11), KINK-1 (SC-naïve n = 6; COVID-19 n = 7) and MMG-11 (SC-naïve n = 4; COVID-19 n = 4) for 2h and then stimulated with zymosan (4h) and nigericin (2h) as indicated. For statistical analysis, two-way ANOVA with tukey post hoc test was used.
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Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for MDGI (green in E and F), and CD31 (red in E and F). Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). Arrows in F point to the MDGI-expressing angiotropic tumour cells. Scale bar: 50 µm.
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(E) Ratio of mice with and without lung metastasis determined by H&E tissue staining. N = 8 mice (shC, PBS), N = 7 mice (shC, AMD), N = 7 mice (shTNC, PBS), N = 7 mice (shTNC, AMD). p < 0,0001, Chi square test. Fisher's exact test was used to compare the conditions pairwise *, p < 0,05, *** p < 0,001. p = 0,0109 (shC/PBS versus shTNC/PBS), p = 0.0001 (shC/AMD versus shTNC/AMD).
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B Relative ferrireductase activities were measured in the cells described in (A). *P = 0.0003
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Loss of interaction between Frizzled 4 (FZD4) and ß-Arrestin 2 (ARRB2) in Grk2-/- NIH3T3 cells. (E) Proximity ligation assay (PLA) between expressed FZD4-GFP and ARRB2-Flag showed significantly reduced proximity events between the two proteins in Grk2-/- cells. White dashed lines outline the transfected cells. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cells are indicated. Scale bars, 10 µm.
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(A) HepG2 cells were transfected with control or ING5, or treated with siRNAs against JFK. Soluble chromatin was prepared and qChIP analysis was performed on the selected promoters using the indicated antibodies. Histone H3 was detected as an internal control. Error bars represent mean ± SD for triplicate experiments (*p < 0.05, paired two-tailed Student's t-test).
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(A) Scheme of the differentiation protocol for hNES cells. The AF22 cells (NES cells derived from induced pluripotent stem cells) were infected at day 2 with TET-on PBX1 lentiviral particles, and doxycycline (DOX) treatment started the day after.
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I) Knockdown of Fasn1 in glial cells rescues NB EdU incorporation defects, caused by glial Hh.N.EGFP overexpression, quantified in (I) (n=15, 13 brain lobes).
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A Western blot analysis probing the localisation and status of plasmid-encoded N-terminally V5-tagged wild-type (WT) or modified (P300A) HybA in S. sonneiΔrhom7ΔhybB with wild-type (+) or inactive (SAHA) GlpG expressed from pUC19. Whole cell lysate (Whole cell.), Sol (Sol.), Mem (detergent-solubilised, Mem.), and the Aggregate (Agg.) fractions are shown. HybA that is uncleaved, cleaved only by GlpG, and further degraded post GlpG cleavage is marked by black, red, and green arrows, respectively.
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(K) Model. Cytosolic peroxiredoxins protect the mitochondrial matrix from internal (mitochondrial) and external H2O2.
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A Layer V cortex explants from E18.5 NB-3+/+ and NB-3−/−mice were cultured in medium supplemented with either Fc or NB-3-Fc (50 μg/ml).B Quantification of neurite outgrowth in (A). n.s., not significant, **p < 0.01; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from more than 50 layer V cortical explants from 5 independent experiments in each group.
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F. Fluorescence micrographs of β'-COPVN•Dsl3pVCcells carrying the myo2-66 defect. COPI•Dsl BiFC, as well as ER or Golgi markers were analyzed. While the BiFC spots were dispersed in these mutants, they retained their association with ER and Golgi. Scale bar 5 μm.
|
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CDF1 (G) and FKF1 (H) mRNA simulations in LDs, from WT (as in C, D) and the prr9;7 mutant (dashed blue-green line).
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D PD-L1 transcript levels in U2OS mock (black) and polyclonal MLLT6 knockout lines (grey) treated with and without IFN-γ normalized to expression in mock U2OS cells without IFN-γ treatment (ANOVA; **p < 0.01; n=4 biological replicates; df=12).
|
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(a) (left) Representative confocal fluorescence microscopy image showing methanol fixed HeLa cell transfected with GFP-Rab34 and stained for LAMP1. Linescan analysis shows relative intensity of GFP and LAMP1 signals. Scale bar = 10μm.
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K DHHC2_C128A-HA mutant protein could be palmitoylated by the co-transfected DHHC2WT-Myc, but not DHHC2_C128A-Myc in HEK293T cells. Representative of two independent repeats.
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B-D Confocal images of early Stage 12 embryos from the control and lines expressing RNAis against (B) porthos, (C) GR/HPR or (D) LKR/SDH, specifically in macrophages (red). Germband edge: dotted white line. Data information: Scale bar: 50µm
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(D) Overlay of the aromatic parts of different spectra for intracellular identification of OMP. The singulet of the H5 proton and the dublets of the H8 and H10 protons can be identified when the medium and the cell spectra are compared to those without OMP treatment. Abbreviations: His - histidine, Tyr - tyrosine, Phe - phenylalanine, Leu, Ile, Val - Leucine, Isoleucine, Valine. Ala - Alanine. Glu - Glutamate, Gln - Glutamine, PC - phosphatidylcholine, Cho - Choline, GPC - glycerophosphocholine, Tau - taurine, Scyllo - scylloinositole.
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Confocal images showing MT network in WT, akap KO, c5rap2 KO, pcnt KO and pc-ak-2KO RPE-1 cells fixed and stained with antibodies against α-tubulin (left) or EB1 (right). The red and yellow lines indicate the contour of cells.
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In silico assessment of N-termini of human disulfide relay substrates. Approximately 40-50 substrates of the human disulfide relay have been described (Habich et al., 2019b). Of those, 13 contain conserved DPP8/9 processing sites at their N-terminus. For COX17 and NDUFA8, the predicted neo-N-termini 'GL-' (COX17) and 'GI-' (NDUFA8) after DPP9 processing were identified in the mouse N-terminome (Calvo et al., 2017). Logo plots represent alignments over163 different species for AK2 and 11 different species for all other proteins. For many disulfide relay substrates, the cleavage sites for DPP8/9 are conserved.
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(B) Wt neurons were kept in culture until DIV10, then the cells were treated with NMDA (50 µM), or vehicle for 30 minutes. Cell surface proteins were labeled with biotin and enriched by streptavidin pull-down. The biotinylated proteins were detected by immunoblotting. Total lysates were analyzed to compare mNrCAMs surface/lysate levels. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (** p < 0.01, n = 6). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.
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J, K, Respiratory control ratio (RCR)(State 3/State4) (J) and P/O ratio (K) in isolated mitochondria from adult fish (male and females Δ1 n=4, and same number for their respective controls) with the indicated substrates.
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E) Bar plot showing the dependence of dwell time on N for wild type Ago2. The dashed red line indicates the observation time limit (300 s), which is constrained by photobleaching.For all bar plots (E, G and I) error bars are the SD of three independent experiments carried out on different days.
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E qRT-PCR analysis show Hh and proliferation target mRNA expression levels. For qRT-PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT). Data show the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.
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Caspase activity of HCT116KO following different treatments as indicated. ANGR66A, ANG receptor binding site variant; ANGK40I, ANG enzymatic activity null variant; BAY, inhibitor of TNF-α-induced κB kinase (IKK) phosphorylation
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(A) Representative images for the time course of PKCα nuclear translocation, subsequent nuclear envelope rupture, and DNA release in human dPMNs exposed to 50 nM PMA for 5, 15, 30, 60, and 180 min, then stained concomitantly for DNA (DAPI), nuclear lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), and PKCα (primary anti-human PKCα, and PE-labeled secondary antibody), following by confocal fluorescent microscopy analysis. White-empty arrows indicate cytoplasmic distribution of PKCα at 5, 15, and 30 min, yellow arrows indicate site of discontinuity/rupture of nuclear envelope at 60 min, light blue arrows display the sites of nuclear envelope rupture and chromatin release at 180 min (A). Scale bars, 20 μm. The time course started 5 min after PMA stimulation in order to allow the adherence of neutrophils on the bottom of dish for immunocytostaining.
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A, B Virus titers in the blood from mice (n=5) fed an NSD (0.45% NaCl) or HSD (4% NaCl) for 30 days (A) or 7 days (B) and then intraperitoneally infected with VSV (1x108 PFU per gram body, 48 hrs) were analyzed by the TCID50 assay. Data information: Data represent mean and SEM of five biological replicates For all statistical testing, p values were calculated using two-tailed unpaired Student's t-test. NS, not significant (p > 0.05). *p < 0.05, **p < 0.01 and ***p < 0.001.
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H, The specific differentially methylated regions (DMR) at the Sirt6 locus were analyzed by PCR amplification and sequencing of bisulphite-modified DNA. Ten independent clones were sequenced per condition. The quantification of methylated (filled circles) vs. unmethylated (empty circles) CpGs is shown in the histogram in the indicated conditions.
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A: Molecular characterization by PCR using established markers: MSP2 IC1, GLURP, MSP1 K1, MSP2 FC27, MSP1 R033, and MSP1 MAD20. B: Markers as in A as well as the sizes for the three parasite strains.
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E. Lipids tolerance test after oral gavage of olive oil to male mice after 9 weeks in HFD and receiving daily CRT0066101 or control. n=6 for control and n=5 for CRT0066101.
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Whole mount images of colon samples from 7-month old mice with indicated genotypes (G). Quantification on density of colon crypts (H) (n=5 mice per group).
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(E, F) Western blot (E) and quantification (F) of COXI in homogenates from spinal cords. Protein levels are normalized by β-actin. Results are expressed as mean ± SEM and relative to Non Tg at 30 days; n=6 mice (3 males and 3 females). *p= 0.017 (Non Tg vs. SOD1-G93A at 120 days) by paired Friedman's test with Dunn's correction. No other statistically significant differences were found (paired Friedman's test with Dunn's correction at 30 and 60 days and paired one-way ANOVA with Tukey's correction at 90 days). COX1 is decreased in SOD1-G93A spinal cord relative to Non Tg at 120 days.
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(D) Molecular interaction between TSC2 and Rheb in the presence or absence of lactate in HA-Rheb- and/or COUP-TFII-overexpressing HCT116 cells treated with 25 nM trametinib for 24 h.
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(C) The amount of fibrils taken up by CAD cells exposed to 1µM fibrils, of fibrils remaining in the cells and exported into the medium after 24h incubation were quantified by a filter trap assay. Data are mean ± s.d (n=3 independent measurements, filtered in duplicate). The standard fluorescence curve for increasing ATTO550-α-synucleinfibrils concentrations is given.
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C. Patient 3, 3 weeks old. Sagittal T1 weighted image (I) demonstrates a very thin severely hypoplastic remnant of corpus callosum (short arrow), a short cingulate sulcus (long arrow) and slender pons. FLAIR image (II) reveals faintly increased signal (short arrow) in the posterior limb of internal capsule (PLIC). There is lack of myelin maturation in the PLIC (short arrows) on T1-IR (III) and T2 (IV) weighted axial images. Focal widening of the interhemispheric CSF space (arrow) on T2 weighted image (IV) reflects absence of rostral fiber tracts). Cingulate gyrus is present (long arrow). No traversing callosal fibers are shown at this level (I).
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A In vitro pull-down assay showing the interaction of NMT1 and EGR2. Recombinant GST-NMT1 was incubated with immunoprecipated MBP-His-EGR2 or MBP-His at 4°C for 2 h. Anti-GST and anti-MBP were used to detect NMT1, EGR2 and MBP-His, respectively.
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E - J Localization of ubiquitin, LC3 and p62 in FBXO2 KO cells. HeLa WT or FBXO2-KO cells were infected with WT GAS for 4 h, fixed, and immunostained for ubiquitin (FK2: magenta) or LC3 (magenta) or p62 (magenta). Representative confocal images (E, G, and I) and percentages of cells with ubiquitin, LC3, or p62 -positive GAS (F, H, and J).
|
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The syntheses of the ligands PR-924, 9, 14, 16, 17 and 18 have been described previously (de Bruin et al, 2014). All ligands share the peptide scaffold shown in the upper left corner with P1, P2 and P3 side chains and an N-cap; they are equipped with a l-Ala (stereochemistry (S)) or d-Ala (stereochemistry (R)) residue at P3 (red) and either an N-acylmorpholine or a 3-methyl-1H-indene group (green). Furthermore, compounds differ in their P2 residue (either methoxytyrosine or tryptophane; blue). Inhibitor 9, also termed LU-015i, is endowed with an exceptional cyclohexyl residue (gray), which is associated with increased selectivity for human β5i.
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Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti-CD3-stimulated thymocytes isolated from WT and Cd4cre A1fl/fl mice. The expression of GAPDH is shown as a reference. Representative images are shown (left panel). The band intensity of p-ZAP70 and p-PLCγ1 was normalized to that of total ZAP70 and PLCγ1, respectively
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A Body weight measured in female and male animals at 3, 6 and 12 months of age. Data are presented as mean ± SEM (n = 5 per group)
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Subcellular protein fractionation of several ER resident proteins in A549 cells treated with the indicated concentrations of Tm or Tg using differential centrifugation protocol. Quantification of the subcellular protein fractionation of several ER endogenous proteins in A549 as in (G). Biological triplicates, mean ± SD calculated using Prism 9 (GraphPad).
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(A) Single cells were isolated from Drosophila embryos 14-14.5h after egg deposition (AED). Isolated cells were processed through a 10x Genomics instrument. After sequencing the resulting libraries, the reads were aligned and processed using the standard pipeline from 10x Genomics. The single-cell gene expression profiles were used to map the cells to known cell types by comparing against the available in situ hybridization patterns from the Berkeley Drosophila Genome Project.
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(C and D) MCF10A cells were treated for 30 minutes with U0126 (a MEK inhibitor), or with no agent, and thereafter EGF was added (10 ng/ml) and incubated with cells for additional 30 minutes (C). Protein extracts were used to assess levels of phosphorylated ERK (C).
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B Strategy for identifying suppression by standing variation. A temperature sensitive (TS) allele collection of ~1,100 partial loss-of-function mutants in the reference background (yellow) was crossed to the 10 wild yeast strains with potential suppressor alleles (blue), to produce large segregant populations selected to carry the TS allele. Each cross was performed in one or two biological and four technical replicates. The fitness of the resulting ~70,000 populations was measured at 26°C (permissive temperature) and 34°C (restrictive temperature). A subset of 38 candidate suppression events were used in bulk segregant analysis for linkage mapping of causal loci that display selection at restrictive temperature (black) but not permissive temperature (grey). D
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GB2 cells infected with the indicated lentiviruses were intracranially transplanted into immunocompromised mice. After 8 weeks, mice (3 to 5 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA were quantified by qRT-PCR.
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Concentrations of IFNβ, measured by ELISA, in the culturing media of MCF7 and T47D cells 48 hours after siRNA transfections. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC, siLMNA: siRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD.
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Co-application of BTX and recAPPsα significantly inhibits synaptic plasticity in cDKO mice. After 1 hour pre-incubation of acute slices with 10 nM recAPPsα or 10 nM recAPPsα and 10 nM BTX fEPSPs were recorded. Hippocampal acute slices treated with 10 nM BTX and recAPPsα revealed significantly impaired induction (t20-25) and maintenance (t75-80) of LTP in comparison to slices of cDKO mice recorded in the presence of recAPPsα alone.
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Intraperitoneal ITT after 20 weeks of high-fat diet intervention. Insulin was adjusted to 1 mU per g of body weight (n = 11-13 animals per group). Area under the curve determined from (D) (n = 11-13 animals per group).
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A Graphs show the quantification of lamin B1 intensity and circularity. N = 3. An average of 12 nuclei was examined in each culture. Data are expressed as a percentage of controls. Bars represent the mean ± S.E.M. *P < 0.05 and **P < 0.01 as compared to mApple-C1 control neurons (two-tailed unpaired Student's t test). Exact P values are reported in Appendix Table S3.
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(a) IL-8 enzyme-linked immunosorbent assay (ELISA) on U2OS cells starved of glutamine or treated with BPTES (10 μM) for 24 h.
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B. Diagram representing the same scenario as in A., but here MSH2 dependent recognition of the mismatch inhibits progression of recombination. This triggers feedback signaling that recruits SPO11-1 complexes to this region and increases meiotic DSB and crossover frequency.
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(C) Probability density distribution for HP2 opening in WT L448A or L448T EAAT1 (GltPh A360); the shaded area indicates closed states.
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Immunostaining of phospho-cMet in the same tumours grown in Rag2-/- mice after treatment with Tepotinib or vehicle control. Scale bar 200 µm.
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PBMCs and plasma samples were collected from SARS-CoV-2 infected individuals at time of hospitalization (D1) and after 5 days (D5). C-D: Percentage of pDCs (C) and mDCs (D) at D1 and D5 matched samples from all patients independent of symptom duration (n=93). Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Wilcoxon matched pairs signed rank test *<p0.05, **<p0.01 ***<p0.001, ns= not significant.
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I. Quantification of the proportion of CD34+α6+ cells that maintain their fate over time (experiments with both EGFP+ or EGFP¯ labeled cells are indicated).
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(G) Left, representative bioluminescence images of lung metastasis in mice after tail vein injection of 22Rv1-Luc cells with different ESM1 expression patterns. Scale bar: 0.5 cm. Right, quantification of the lung bioluminescence by photons/ seconds.
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COs transfected with micro-RNAs targeting ECE2 (KD) or scrambled negative control (CTRL) and GFP and analysed 7 days later reveal an increase in ectopic neurons upon ECE2 KD (Transfected cells are shown in green, NEUN+ neuronal nuclei in magenta. d=day. Scalebar=25 µm). Ventricle-like lumen in COs is marked as V'.
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D-F Relapse-free survival (RFS) of breast cancer patients. Data were downloaded from the Kaplan-Meier plotter
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E Visualization of the interaction between PBL27 and MAPKKK5K375M by BiFC analysis in Arabidopsis protoplasts. FLS-associated RLCK BIK1 was also used for this assay. Venus fluorescence indicates interaction between PBL27 and MAPKKK5K375M. Scale bar=10µm.
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Oculomotor complex of POLG patient with progressive ophthalmoplegia; midbrain level. Hematoxylin-eosin staining. The whole area shows spongiotic degeneration. Scale bar 50 µm.
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G. Hierarchical clustering of the 1.000 most variable probes within the dataset of embryonic development of yolk sac-derived tissue MΦ (EMP: erythromyeloid precursor, A1/A2: yolk sacMΦ progenitors, embryonic microglia (Emb F4/80 MG), embryonic Kupffer cells (Emb F4/80liver), embryonic (Emb) F4/80kidney cells. Heat map displays z-transformed log2-expression values from red to blue via white.
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The production of (B) TNF‐α, (C) IL‐6 and (D) IL‐1β was measured in culture supernatants using Milliplex technology. Box plots depicting median values and quartiles are representative of n = 9 experiments.
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C Ratio (488/440) image of BCECF fluorescence in the roots of MdCAX3-suppressed Mx. Scale bars: 100 μm.
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(A) Western blot analysis of NDUFS3 protein levels in homogenates from quadriceps muscle of 15-days old (upper panel) and 1-month old animals (lower panel).
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a-f, Histological analyses of Atg7flox/+; nestin-Cre (left) and Atg7flox/flox; nestin-Cre (right) cerebral cortex at P56. Cryosections were stained with HE (a-d) or immunostained for the glial marker GFAP (e, f). Boxed areas in a and b are magnified in c and d, respectively. Arrows in c point to large pyramidal neurons in the cerebral cortex.
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(L) Social interaction behavior is impaired in mH2A1.2 KO mice. WT mice spent similar time in the chamber with the stranger 1 or stranger 2 mice, while WT mice spent more time in the chamber with stranger 2 than with stranger 1. (M) Close social interaction is affected by mH2A1.2 deletion. KO mice did not spend more time in close interaction with stranger 2 than with stranger1 mice. Date information: All mice were male, 8-12 weeks old. WT, n=12 mice; KO, n=12 mice. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**) or P< 0.001(***). n.s., not significant. Analysis was performed by researcher blinded to the experimental conditions.
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E 125I‐labeled Y‐GRIp68-78 (0.46 nM) bound specifically to Col‐0membrane fractions (light gray bars), the binding was significantly reduced in prk5‐1 and prk5‐2 plants. Excess of non‐radioactive Y‐GRIp65-84 (10 μM) reduced binding to background levels (dark gray bars; all bars show the average of four samples, triangles show individual data points).
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(B) ClustalW sequence alignment for Spindly orthologues. The region encompasses the Polo-dependent phosphorylation site identified in Drosophila Spindly (red box). Asterisk highlights a conserved residue identified in silico by the Eukaryotic Linear Motif (ELM) resource as a putative Polo-dependent phosphorylation site in human Spindly. Sequence alignment was coloured according to clustal colour scheme in Jalview (www.jalview.org, v2.10.5). Abbreviations: h, Homo sapiens; m, Mus musculus; x, Xenopus laevis; d, Drosophila melanogaster. Data information: Scale bar: 5 μm.
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D) Progression of the elongation ratio (red - manual, black - PIV) of nests Note the initial fast decay on the elongated shape of the nests.
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(B) Contour plot of the HP distance vs. the R479-E406 (GltPh Q318, R397) distance distribution. Gray lines indicate thresholds for the HP2-closed and salt bridge-formed states.
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Analysis of apoptotic cells at the indicated time points. HLEC cells were treated with vehicle (V) or 0.5 µg/ml BO-110 (BO) for the indicated time points. Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.
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Male Raptor flox/flox mice (8 weeks) were fed a HFD for 12 weeks and injected with AAV8- GFP or AAV8-shDOCK5 or AAV8-shDOCK5 + AAV8-Cre via the tail vein. Total and phosphorylated InsR, IRS-1Ser1101 and Akt in the liver.
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(E) MDA-MB-231 cells were transfected with siRNAs targeting CFL1 or a control siRNA in the setting of control or PLCβ1 overexpression were subjected to the migration assay. N = 5 inserts/group.
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(B) Seed sets (mean ± SD, n = 5) of WT, asy1, pch2, asy1 pch2, ASY1T142V;T184V:GFP (asy1) and ASY1T142V;T184V:GFP (asy1 pch2) plants. Asteriks indicate significant differences (two-tailed t-test, P < 0.01) and ns depicts no significant difference. Scale bar: 2 mm.
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(L and M) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, and RFP-LC3 for 30 hr were loaded with 10 μM PI(3) P for 1 hr in complete medium, and then fixed and imaged on confocal microscope. Bar, 10 μm. Quantification of cells (percentage of total) showing more than 10 RFP-LC3 vesicles in the different conditions from (J) is shown in (M); n = 200 cells (mean ± SEM). See also Figure S5.
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f, In-gel fluorescence assay (top panel) visualizing Alexa Fluor 647-labelled myristoylated ARF1-His isolated from bacteria either not expressing (lane 1) or expressing (lane 2) NMTp. Bacterial cell lysates treated with recombinant IpaJ or IpaJ C64A as indicated. The expression levels of ARF1-His were determined by Coomassie blue stain.
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A) Differential interference contrast (DIC) image of cortical neurons in culture. The arrowhead points to a neuron expressing Ub-R-GFP. B) GFP fluorescence before and after exposure to lactacystin (10µM).
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A Immunostaining for microtubules after transient expression of hTau and hP301L. Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.
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C) Larval brains at 0 h ALH from grh-Gal4; UAS-CD8-GFP were labeled with γ-tubulin (γ-tub), Asl, and GFP and imaged under super-resolution microscopy. D) Larval brains at 0 h ALH from grh-Gal4; UAS-CD8-GFP were labeled with Centrosomin (CNN), Asl and GFP and imaged under spinning disc super-resolution microscopy.
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(E) Confocal images of formaldehyde fixed, co-isogenic GFP+ wild type and mutant S. flexneri strains harboring rfb regions of E. coli serotypes O8 and O25 after 60 min of incubation time in the presence of 10 µM Alexa-Fluor647-hGBP1F and 2 mM GTP.
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(F) The potential for glucose uptake by HSCs obtained from untreated or 5-FU-treated mice was estimated using 2-NBDG.
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E. Gene set analysis based on NanoString advanced analysis R-script included in the Neuropathology panel.
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(G) Hippocampal neurons were transfected with ErbB4 containing a luminal HA-tag (HA/T-ErbB4) and shCtrl or shTDP (DIV9+3). Surface HA/T-ErbB4 was stained in living cells and intracellular HA/T-ErbB4 was stained after fixation and permeabilization with HA-antibodies. (H) Quantification of HA/T-ErbB4 surface levels. At least 10 images per condition per experiment were analyzed in three independent experiments. Scale bar represents 50 µm.
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C) Human colon organoids cultured in full medium (FM) condition (upper panel) or FM treated with IFN-γ (lower panel). Left: bright field images of 7-day cultured human colon organoids, scale bar: 100 µm. Right: Immunofluorescence for KRT20 (green; note increased expression upon IFN-γ treatment). Scale bar: 25 µm.
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(C) Representative histograms of c-Maf and GATA3 expression in ILC2s from adult PBMC or cord blood cells and percentages of c-Maf+ and GATA3+ ILC2s from adult PBMC or cord blood (n=5 different donors; T test, ****p<0.0001).
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(C) Quantification of activated microglia within 30 µm from plaque borders. Top: Numbers of Iba1-positive microglia were normalised to the size of the nearest 4G8-positive plaque and quantified in male (n=10) and female (n=8) APP23 mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.0576. Bottom: Histogram representing Clec7a staining intensity within plaque-associated Iba1-positive microglia in male (n=10) and female (n=8) APP23 mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test with Bonferroni correction for each single bin, p=N.S.. Right: representative images, scale bar = 40 µm. Data points were taken from graphs analysing APP23p40+/+ against APP23p40-/- mice.
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(A) RCC1.24‐NeoR (endogenous), and RCC1.24 cells to which NeoR protein had been added (exogenous), were treated with different concentrations of 3‐MA and cell surface presentation of the NeoR‐derived peptide on HLA‐DP3 monitored with the T cell clone 20-4/A4‐specific T cell clone 20-4/A4. Inhibition of autophagy by 3‐MA causes an almost complete inhibition of endogenous antigen presentation, while presentation of exogenous NeoR is not affected.
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G Quantification of ciliary localisation of GFP-RAB35 in hTERT-RPE1 cells stably expressing GFP-RAB35 treated Data are mean ± S.E.M. of 5 independent experiments. Statistical significance according to ANOVA followed by Bonferroni post-hoc test (* P < 0.05, *** P < 0.001; P-values: Neg vs. DENND1B P < 0.0001, Neg vs. TBC1D10A P = 0.0322).
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Cryo-EM density map colored by each subunit. Bottom panel is rotated 90° relative to the top panel. Colored as in A, the refined atomic model of the TRAPPIII complex.
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B and C) Confocal images of K562 cells transiently expressing MplmOrange2. Cells in (B) are untreated. Cells in (C) were stimulated 60' with Tpo, washed and chased for 15 min before imaging.
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(A) Single confocal sections of HeLa cells infected with GFP-expressing S. Typhimurium strains (blue) for 10 h, fixed and immunolabelled for LAMP1 (green) and ubiquitin (Ub, red) (false coloured, scale bars, 5 µm). The far right panels show merged images of LAMP1, ubiquitin and Salmonella. Arrows indicate SCV-associated ubiquitinated aggregates.
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(B) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced (donor), non-transduced (receiver) neurons. Four groups were excluded due to very high GFP transduction rate (GFP-negative donor) and very low GFP transmission rate (GFP-positive receiver with IgG and anti-GA) and complete prevention of GA-RFP transmission of anti-GA immunodepletion (GA-GFP receiver with anti-GA). n=3 biological replicates. In total 280 donor GFP, 284 receiver GFP with IgG, 317 receiver GFP with anti-GA, 277 donor GA175-GFP, 294 receiver GA175-GFP with IgG, 311 receiver GA175-GFP with anti-GA cells were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001.
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G Cells that always enrich HXT2 mRNA in the bud have a growth advantage in the first hours when coming from quiescence compared to WT or cells that only express HXT2.
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D Unsupervised cluster analysis of the 102 FDA-approved drugs based on the correlation structure of the GI50 levels. The result is shown as a symmetric heat map with positive associations depicted in yellow and negative associations shown in blue.
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NADH-dependent respiration (either aerobic or anaerobic/nitrate respiration) allows cells to keep and active TCA cycle and proper acetate catabolism by oxidizing NADH. Free fatty acids accumulate under NADH-deficient respiration and repress the expression of SaeRSPQ, which regulates the production of virulence factors and biofilm formation.
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Western blot (WB) image of human LECs that were either kept unstretched or stretched for 30 minutes, and used for immunoprecipitation (IP) of HA-tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates Quantification of the ILK protein amount in IP lysates from LECs with (+) or without (-) mechanical stretch; normalised to the respective amount of HA-tagged β1 integrin (n = 3 (unstretched) or n = 5 (stretched) independent stretch chambers
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Orthotopic xenograft and primary MPNSTs exhibited similar immunohistochemical features. A representative immunostained section of vimentin, CD34, S100, and Ki-67 is shown for primary tumors (PT) and orthotopic tumors (OT) from patientsMPNST-NF1-001 and MPNST-SP-002. Positive antibody signals are shown in brown, and the hematoxylin counterstain in blue. Main panels show pictures at high magnification (400×); inset pictures show mitotic cells present in these tumors.
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