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(D) mRNA levels measured by RT-qPCR in G1 synchronized cells in the indicated strains. Expression is normalized to either snR13 or FIG2. Data are expressed as a ratio of Rpb3-TAP/WT in percentage.
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A Yeast two-hybrid (Y2H) assays showing the interaction between SEU and SCR. The yeast transformants were plated on synthetic defined (SD) media lacking Leu and Trp (SD/-2) or lacking Ade, His, Leu, and Trp (SD/-4) to assess protein-protein interactions. AD, GAL4 activation domain; BD, GAL4 DNA-binding domain.
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F) The CUX1 gene (cut-like homeobox 1) contains 2 interleaved clusters of MXEs (clusters 1 and 2) and 2 standard clusters each with two MXEs (clusters 3 and 4). The exon 3 and exon 4 variants each are orthologous exons. The exon 4 variants are mutually exclusive (cluster 2). Exon 3a is a differentially included exon and only spliced together with exon 4a. The exons 3b, 3c, 3d and 3e are part of a cluster of four MXEs (cluster 1) and are only spliced together with exon 4b (Appendix Fig. S16 and S17). Novel exons are labelled with an asterisk.
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C Duration of the short telomere-induced crisis for the clones analysed in A) and B). The crisis period was determined as the number of days the cell population stayed arrested without notable increase in cell density. The average crisis period is plotted for each genotype. The error bars are SDs of n independent clones: est2∆ (n=10), est2∆ shs1∆ (n=16), est1∆ (n=6) and est1∆ bud6∆ (n=7) . P values are from two-tailed Student's tests (*p=0.014).
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Sorting strategy for SBROS cells in G1 or S phase and into the four following subpopulations: SOX2 high & OCT4 high (SHOH), SOX2 high & OCT4 low (SHOL), SOX2 low & OCT4 high (SLOH) or SOX2 low & OCT4 low (SLOL).
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(G) Blood parasitemia (mean +/- SEM) at day 4 and 6 pi in Karma mice treated (open circles) or not (black circles) with DT. Day 4, P = 0.022 ; Day 6, P = 0.77 by multiple unpaired t-tests without assuming consistent SD. C,E,F,G: No DT group, N = 5 mice ; + DT group, N = 6 mice. Representative of 3 independent experiments.
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D. Mutations in H149, N152 and D160 abolish the ability of MavQ to convert PtdIns into PtdIns3P.
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D Electron micrograph illustrating organelle masking of MAM interfaces; scale bar= 500nm. Data information: er, endoplasmic reticulum; m, mitochondria.
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Recycled levels of BACE1 after 20 min of HA antibody pulse in HA-BACE1-mCherry-transfected WT and KO neurons, co-expressing either eGFP-RAB4 or eGFP-RAB4-S22N pWTRAB4 vs pWTRAB4S22N>0.999, 34-38 neurons, N=4 biological replicates). Scale bar: 5 µm. Data information: Experimental designs are depicted in the top panels , where analyzed BACE1 fraction is marked with the red square.
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I, J and K: kymographs for GFP-myo intensity, T, and edge velocity along the cell outlines for the second cell of A.
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B. Growth of the reconstructed LysP T33F mutant compared to wild type cells transformed with a Non-Target gRNA. n=3. C. Growth of the reconstructed LysP Q219I mutant compared to wild type cells transformed with a Non-Target gRNA. n=3
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B Gel images from RT-PCR show purity of the sorted nuclei from a representative PD sample. Nuclear RNA was isolated, cDNA was generated and pre-amplified (see Materials and Methods for details) before target-specific PCR. Neuron specific genes (NeuN, synaptophysin) and astrocyte specific gene (GFAP), α-synuclein, and GAPDH were amplified from the isolated nuclei.
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(f) Quantification of MCF10A E7+Bcl2 colony formation with Atg5 knockdown with two separate siRNAs. Data represent mean±s.e.m.from three independent experiments; *P0.001. (g) Representative wells for data in f. The insets show higher magnifications. See also Supplementary Movie S6.
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(F, G) Working model for hTLR4 activation enhanced by VLCFA-GM3 species (F) and reduced by LCFA-GM3 (G). Basic residues contributing to GM3 recognition are colored in blue. Residues of dimer interface are colored in red.
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A. Expression of CSR1 to 35 was evaluated by qRT-PCR in RNA isolated from control cells or cells infected with a moi 0.3 of HCV for 6 days. GAPDH levels were also measured and used as a reference. Fold increase of each CSR in infected versus control cells is indicated at the top of each bar. A line has been drawn to mark the position of 7 fold, our arbitrary cut-off value. Black bars, candidates expressed to good levels that increase more than 7 fold; white bars, candidates expressed poorly according to the parameters described in Appendix Table S.2; grey bars, candidates expressed to good levels induced less than 7 fold. CSR28 bar was shaded to highlight that this candidate was not evaluated further.
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(c, d) Parkin-directed mitophagy specifically involves linkage of ubiquitin through lysines 27 and 63. HeLa cells were transfected with EGFP-Parkin wild-type and ubiquitin lysine variants and treated with CCCP before immunostaining with anti-HtrA2/Omi, as a mitochondrial marker (pseudo coloured; blue) and anti-His (red) to detect ectopic ubiquitin. Parkin was visualized by its fusion to EGFP (green). (c) Note that only wild-type, K27 and K63 6×His-tagged ubiquitin variants are colocalized with condensed mitochondria and EGFP-Parkin after 6 h of CCCP treatment. All other ubiquitin mutants interfere with Parkin translocation to mitochondria. (d) Only co-expression of K27 or K63 ubiquitin recapitulates mitochondrial clearance similarly to 6×His-tagged wild-type ubiquitin after 24 h of CCCP treatment. Representative images are shown. Scale bars, 10 μm. Immunostaining of untreated cells and all other ubiquitin variant combinations tested are shown in Supplementary Information, Fig. S4a and b, c, respectively. WT, wild-type; PolyUb, poly-ubiquitin.
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Dot plots depicting the abundance of neuroblasts (DCX+ Ki67+ or DCX+ Ki67-) in the SEZ 7 days after aCSF or AntimiR204 injection.
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(B) Time-lapse observation of the dynamics of NuMA and chromosomes. Centrinone-treated HeLa cells expressing mCover-NuMA were observed with a 63× objective. Magenta and green represent SiR-DNA and mClover-NuMA, respectively. Arrowheads and two-way arrows indicate the assembling NuMA after NEBD and bipolarity, respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD.
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GFP-tagged DIA1(WT), DIA1(R1204X), and DIA1(M1190D) were transfected into XTC cells. XTC cells were spread on the PLL-coated coverslips and time-lapse images were acquired every 300 ms, illuminating the restricted areas near the cell edges, by using a single-molecule speckle microscope. Representative images are shown for DIA1(WT) (A, Movie EV5), DIA1(R1204X) (B, Movie EV6), and DIA1(M1190D) (C, Movie EV7). Circles indicate speckles showing directional movement over a distance of several micrometers. Trajectories of these speckles are also illustrated, and crosses were plotted at the end of the directional movement. Speckles showing directional movement were frequently observed in cells expressing GFP-DIA1(R1204X) (B), whereas such speckles were scarce in cells expressing GFP-DIA1(WT) (A). However, the density of these speckles in GFP-DIA1(R1204X)-expressing cells appeared lower than that in GFP-DIA1(M1190D)-expressing cells, which is considered a fully active mutant (C). To evaluate the frequency of speckles indicating directional movement, we performed a quantitative analysis (D). The number of speckles showing directional movement during 3 continuous planes was counted and were normalized by the fluorescent intensity of each cell, which corresponds to the expression level of the GFP-tagged protein. *P= 0.0288 and **P= 0.0002 Bonferroni's post hoc test following one-way ANOVA (WT; n = 5, R1204X; n = 6, M1190D; n = 6). Time is in seconds. Scale bars: 5 µm.
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(1) Tau acetylates β-catenin at K49, which inhibits K49-ubiquitination, Ser45/Thr41/Ser37/Ser33-phosphorylation and proteolysis of β-catenin, leading to β-catenin upregulation. (2 and 3) The increased β-catenin is translocated into the nuclei (2) where it promotes transcription activity and increases expression of bcl2 and survivin leading to cellular anti-apoptosis (3).
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Supernatants from infected cells were collected during the time course in F and assessed for the production of new infectious viral particles using a TCID50 assay on naïve Vero cells. n = 3-6.
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Residue-specific PREamp of Bax (α2-α5) in DMPC/DHPC bicelles with q = 0.6 determined from the lipophilic PRE analysis. The PREamp values were derived from the 16-DSA titration. The plot is colored according to the PREamp values that are proportional to the effects of 16-DSA on the individual residues of Bax (α2-α5). The residues with the least PREamp values in the blue colored areas are in the flexible terminal regions.
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I Both LAMTOR1 KD and TAT-2031 treatment (10 µM) increased ML-SA1-induced TRPML1-GCaMP6m responses in neurons. J Quantification of peak responses as shown in I. N = 6-13 cells from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post hoc analysis *P < 0.05, **P < 0.01, ***P < 0.001, ###P < 0.001, n.s., not significant. traces represent the mean values of each group.
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(c) LDH assay revealed that H2O2 treatment (300 μM; 30 min) resulted in a significant increase in neuronal death 12 h after treatment (n = 8; 2.50 ± 0.12; P 0.001 to control), which was rescued by breaking down H2O2 with catalase (100 U in 10 μl phosphate-buffered saline; n = 4; 1.17 ± 0.02; P = 0.001 to H2O2 group). H2O2-induced neurotoxicity was significantly reduced by TAT-GluN2Bct-CTM (50 μM; applied 60 min before and maintained throughout the experiments; n = 9; 1.56 ± 0.08; P = 0.001 to H2O2 group), but not by TAT-GluN2Bct (50 μM; n = 9; 2.63 ± 0.10; P = 0.105 to H2O2 group) or the NMDAR antagonist APV (1 μM; n = 4; 2.16 ± 0.14; P = 0.169 to H2O2 group). NH4Cl abolished the neuroprotective effect of TAT-GluN2Bct-CTM (n = 4; 2.39 ± 0.27; P = 0.538 compared to H2O2 group). One-way ANOVA, P 0.001, F(6,41) = 26.842. ***,ΔΔΔP 0.001; bars represent relative mean values ± s.e.m. normalized to the saline control (white bar, arbitrarily set as 1). n represents individual experiments from at least 3 separate primary cultures. Full-length blots are presented in Supplementary Figure 9.
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The top panel is a representative RT-PCR electropherogram depicting the inclusion or exclusion LAS1L exon 9 in cells at day 2 as well as cells at day 5 without or with PLB treatment with indicated concentrations, respectively. The quantification of percent spliced-in (PSI) was presented in the bottom bar graph. P-values were calculated by one-way ANOVA followed by Dunnett's test.
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Change in polyA site usage by the R495X mutation. Fold change of polyA site usage by the R495X mutation (R495X/WT) on the relative positions of all mouse coding genes is plotted. The average is shown in blue line. The standard error of the mean is shown in semi-transparent blue shade.
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b, L. monocytogenes c.f.u. from a were determined by plating (means ± s.d., n = 3-5, **P < 0.001 by Student's t-test).
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Distribution of cell length of WT and mutant strains when grown in M9 medium supplemented with amino acids (gray) or with both amino acids and 1mM dTMP (pink). WT, W133V and V75H+I155A strains were grown at 42°C while I91L+W133V and V75H+I91L+I155A mutants were grown at 40°C. 1mM dTMP largely rescues filamentation of mutant strains (* indicates the median cell lengths were significantly different, Mann-Whitney test, p-value <0.001). The central band in the box plots represents the median of the distribution, the box ends represent the 25th and 75th percentile, the whiskers represent the 10th and 90th percentile, while the dots represent the 5th and 95th percentile. Data was usually obtained from 2-3 biological replicates. The number of cells used to derive the boxplot distributions in the different panels range usually between 150 to 300 (please refer to Figure 5 source data for exact number of cells for each dataset).
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Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with wild type B cells with or without 1μg α-GalCer and with or without agnostic anti-CD40. IgM was measured by ELISA of culture supernatants 4 days later.
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B The dimensionless Kratky plots of SAXS profiles of BSP3 in 10 mM Ca2+ (red) and 10 mM EDTA (black).
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G. Network of phosphorylated proteins belonging to "protein localization" in FGF10 clusters, based on STRING, visualized in Cytoscape, and color-coded based on cell components. The squared shape represents phosphorylated proteins found in the database DISEASES. The recycling adaptors TTP and RCP are highlighted in grey.
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G A plasmid encoding the truncated mutant composed of Smad6 amino acids 422-441 (Myc-Smad6(422-441)) or wild-type Smad6 MH2 domain (Myc-Smad6-MH2) or full-length Smad6 (Myc-Smad6) was co-transfected with HA-tagged full-length Smad4 or HA-tagged full-length Pellino-1 plasmid into HEK293 cells, respectively. Cell lysates were immunoprecipitated (IP) with anti-Myc and immunoblotted (IB) with anti-HA or anti-Myc antibody, respectively. The vector, pCS3MTBXA-6xMyc, was co-transfected with HA-Smad4 or HA-Pellino-1 as a negative control. Data are representative of at least three independent experiments.
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B Telomere restriction fragment (TRF) analysis shows increased telomeric length heterogeneities in ATRX-deleted U87-T and the further enhancement following Ad-Cre treatment. Genomic DNAs (EtBr staining, left panel) prepared from control and ATRX-deleted U87-T cells at day 9 post control or Ad-Cre infection (middle panel) were assayed by hybridization with 32P-labelled (TTAGGG)4 probe, followed by re-hybridization with an oligonucleotide probe specific for centromere region (right panel). Genomic DNA from U2OS cells was used as an ALT positive control.
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(b) HeLa were transfected with Ptch1 or Ptch2 together with HA-HttQ74 and the percentage of transfected cells with aggregates was assessed by HA immunofluorescence. P-values were calculated by odds ratio.
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Cd44, PAI1 (also known as Serpine1), and Id1 mRNA expression levels in KRIT1 wt and KRIT1-KO endothelial cells were assessed by quantitative real-time PCR. Where indicated, KRIT1 wt and KRIT1-KO endothelial cells were treated with 100 nM Torin1 or 500 nM rapamycin for 16h. The data are expressed as the mean ± s.e.m. Cd44: *P = 0.02848 (KO ctrl vs. KO Rapa); *P = 0.02605 (KO ctrl vs. KO Tor1). PAI1: *P = 0.04446 (KO ctrl vs. KO Rapa); *P = 0.03996 (KO ctrl vs. KO Tor1). Id1: *P = 0.00266 (KO ctrl vs. KO Rapa); *P = 0.01554 (KO ctrl vs. KO Tor1). n = 3 independent experiments.
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(b) SLC7A11 mRNA levels were decreased upon ATF4 depletion but not NRF2 depletion. HLE cells were transfected with siCtrl, siATF4 or siNRF2 and cultured with DMSO or 6μM Sorafenib for 18 hours. Quantitative RT-PCR was used to determine SLC7A11 mRNA levels. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.
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Human epidermal cells treated as before were challenged for 48h with 20 and 200ng of p24 of HIV-Luc. When indicated cells were pre-treated with 10μM Nevirapine for 30 min before viral challenge. Luciferase activities measured in duplicates for each condition from two (infection with 20 ng P24) or three (infection with 200 ng P24) different skin samples were represented on a graph as means of relative luminescence units (RLU) ± SD.
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(B) μCT analysis of the femurs of 10-week-old control (n = 6), Tet2Rank-/- (n = 5), Tet3Rank-/- (n = 5), Tet2Rank-/-; Tet3aRank+/- (n = 6), Tet2Rank+/-; Tet3aRank-/- (n = 6) and Tet2Rank-/-; Tet3aRank-/- (n = 4) male mice (axial view of the metaphyseal region). (C) Histological analysis of the proximal tibias of 10-week-old control, Tet2Rank-/-, Tet3Rank-/-, Tet2Rank-/-; Tet3aRank+/-, Tet2Rank+/-; Tet3aRank-/- and Tet2Rank-/-; Tet3aRank-/- male mice (TRAP staining [red, osteoclasts]).
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(A) Wild-type and FIP200−/− MEFs were treated with 100 ng/ml rapamycin (rapa) or vehicle (DMSO) for 120 min in the presence or absence of 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody.
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(A, B) SARS-CoV-2 replication shown as infectious virus titers (A) and viral RNA copies (B) in apical washes at 2, 24, 48, and 72h after infection at MOI 0.1. Cultures were either pretreated with 50 ng/ml IFN lambda 1 in the basal compartment two hours prior to the infection (green), at 24 h p.i. or left untreated. The dotted line indicates the lower limit of detection. Error bars represent SEM. N=2. H p.i. = hours post infection.
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D. Clonogenic assay showing that stable expression of wild-type HUWE1, but not of the PIP box mutant or the catalytic mutant, corrects the HU sensitivity of the HUWE1-knockout 293T cells. Shown is the average of 3 independent experiments -/+SD. If not indicated otherwise, the p-values shown specify the statistical significance relative to 293T (for the 0.5mM HU condition). P-values are: 0.0003 (293T vs HUWE1-/-; 0.157 (293T vs HUWE1-/- +WT); 0.0045 (293T vs HUWE1-/- +FF); 0.0006 (293T vs HUWE1-/- +C4341A; 0.047 (HUWE1-/- +WT vs HUWE1-/- +FF).
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c HEK cells transfected with a RlucII-conjugated CXCR4 and an eYFP-β-arrestin 2 construct were stimulated with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD. Control experiments were performed as indicated. Results are given as the net BRET ratio (i.e. ratio of emissions at 535/485 nm minus the ratio of mock cells, n = 5, three technical replicates).
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b, Carboxy-terminal V5immunoblot of full-length (68 kDa) and cleaved (36 kDa) ATG16L1β. Silver stain (bottom gels) depicts total protein. Data represent 3 independent experiments. IB, immunoblot.
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(a) Western blot analysis showed beclin 1 cleavage products (Mr49 K) were not observed in primary neurons exposed to mitophagy-inducing sublethal concentrations of rotenone (250 nM×2 h: Rot low) or STS (100 nM×2 h). However, beclin 1 was cleaved on exposure to a lethal dose of rotenone (1 mM×24 h: Rot high). Veh, vehicle; CTD, C-terminal domain.
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I. Schematic representation of the experimental paradigm. 5µM MB were electroporated. Abbreviations: MB, molecular beacon.
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U2OS cells stably expressing ATF4-reporter were treated as described above for 12 h. The expression and nuclear translocation of ATF4 was assessed by fluorescence microscopy and the nuclear green fluorescence intensity was quantified. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM
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Sub-tomogram average of the IM sub-complex constituting the C- ring, MS-ring and export apparatus. Schematic representation of the sub-tomogram average shown in (A) highlighting the different parts of the complex. Sub-tomogram average of the motors of fully-assembled flagella. Slices through electron cryo-tomograms showing neighboring PL and IM sub-complexes. Dashed-yellow circles highlight the IM sub-complex while dashed-blue arrows highlight the PL sub-complex. Dashed-red lines mark the border between two images used to make a composite image when the PL and IM sub-complexes were found at different levels in the tomogram. Schematic representation of an IM sub-complex in the vicinity of a PL sub-complex. Slices through electron cryo-tomograms of L. pneumophila highlighting the presence of a fully-assembled basal body lacking the hook and the filament. Schematic representation of the basal bodies in (L and M). Central slice through an electron cryo-tomogram of a lysed cell. The dashed-yellow circle highlights the flagellar motor. Enlarged view of the same slice shown in (O). The absence of the C-ring and the export apparatus is highlighted by the dashed-orange arrow. Schematic representation of the complex found in (P).
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F. Co-expression of HA-Parkin with Miro1 does not suppress mitochondrial motility in axons of cortical neurons. Overexpression of Miro1 alone increased the motion time in both directions as well as the relative mitochondrial velocity. Co-transfection of Parkin did not affect significantly the motility parameters. Data are presented as Tukey boxplot. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, n = 689-846 individual mitochondria from 39-44 axons per group pooled from at least from 10 individual dishes from 4 independent experiments, Kruskal-Wallis test
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(G) Model illustrating how mutations in p53 induce loss of function but also gain of new functions via isoform expression. The semi-transparent boxes represent plausible minimal full-length p53 and short p53 isoforms activities.
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Hela cells were co-transfected with the RFP-based TDP-NLS reporter, GFP or GA175-GFP, as well as PSMD11 or empty vector. Image analysis as in (A). n=4 biological replicates. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. 354 GFP and 330 GA175-GFP cells with vector, 367 GFP and 369 GA175-GFP cells with PSMD11 in total were analyzed.
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B The kidneys from control and Lmna-/- mice were stained for acetylated tubulin (green) and AQ1 (a marker for the proximal tubules, red). The insets show the cilia of proximal renal tubule cells.
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(C) Percentage of IFN/TNF-double producing cells among activated CD4 T cells for each peptide. Basal level with MutuDC alone was subtracted. C,D: Data show the mean +/- SEM. Asterisks show statistical significance assessed by paired non-parametric Wilcoxon tests in comparison with OVA peptide. AMA1, P = 0.0098 ; TIM14, P = 0.078 ; ENO, P = 0.016 ; GAPDH.1, P = 0.002 ; MAHRP, P = 0.0039 ; EF1α, P = 0.0039 ; M1, P = 0.016 ; MSP1, P = 0.031 ; LDH, P = 0.062 ; ETRAMP, P = 0.001 ; ATPSYN, P = 0.25 ; END70, P = 0.25 ; PDI, P = 0.16 ; GAPDH.2, P = 0.99 ; pRBC, P = 0.001. N = 6 mice for ATPSYN, END70, PDI, GAPDH.2, N = 11 mice for all other peptides, pooled from 3 replicates.
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B and C. Blood glucose level (B) and plasma insulin level (C) of WT and TRPV2KO mice after 8-week HFD treatment.
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(C) Multidimensional scaling (MDS) was performed and transcriptional regulator co-recruitment was depicted using density plots for regions with increased (a), decreased (b) or unchanged (c) H3K27ac levels in MPH upon acute ERS. The boxed area (d) represents a subset of transcriptional regulators (TR) with a high degree of co-binding in H3K27ac DOWN regions. LIVER-ID TFs are depicted in red.
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(B) Signal intensities of ChIL-seq correlated with the expression levels of genes. The lines indicate the average CPM of each expression group at TSS. The expression groups were assigned with respect to the expression levels (TPM) of genes.
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I, J, K. 35S-radiolabeled recombinant Myc-ATG13 (J) were added to GST, GST-C9orf72S and GST-C9orf72L immobilized on glutathione-coated beads. 35S-radiolabelled recombinant proteins were visualized by phosphoimager (top panels). Coomassie-stained GST, GST-C9orf72S and GST-C9orf72L in the pull-down samples are shown (bottom panels). The identity of the Coomassie protein bands was confirmed by mass spectrometry (# = E. coli DnaK Chaperonin; * = E. coli 60kD Chaperonin; Appendix Fig S2).
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(B) Immunoblot analysis of TFEB shift in ARPE-19 CLN3 KO cells. β-actin immunoblotting were performed as a loading control.
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B ChIP-qPCR assay of H3K4me2 levels of indicated regions at FLC chromatin. C ChIP-qPCR assay of H3K4me3 levels of indicated regions at FLC chromatin. D ChIP-qPCR assay of H3K36me3 levels of indicated regions at FLC chromatin. E ChIP-qPCR assay of H3K27me3 levels of indicated regions at FLC chromatin .Data information: For ChIP analysis, 12-d-old plants were collected and three independent experiments were conducted. Each bar represents the mean ± s.d. of three independent experiments, n=3. The relative abundance was normalized to the input.
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Co-depletion of STN1 and BRCA2 increases chromosome instabilities. U2OS cells with siBRCA2 and/or shSTN1 knockdown were treated with HU (2 mM, 3 h). Representative metaphase images show aberrant chromosomes (red arrows). Boxed areas are amplified and shown at the bottom of images. Scale bars: 20 µm. Two independent knockdown and chromosome spread experiments were performed. N, the number of metaphase spreads analyzed in each sample.
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B. Plasmids containing ELP2 and the four loop mutants were transformed into elp2Δ yeast. The strains were then plated onto -Ura media containing caffeine. The pRS416 vector is used for these experiments.
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Schematic representation of the metabolic approaches used to characterize B16-F10 cells 1H-NMR stands for proton-based nuclear magnetic resonance, GC-MS for gas chromatography coupled to mass spectrometry, OCR for oxygen consumption rate and ECAR for extracellular acidification rate.
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Single-cell expression data in pseudotime of central regulators of naïve pluripotency, epiblast marker genes, glycolytic regulators, and genes with key functions in oxidative metabolism.
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(C) HEK cells transfected with SODG85R‐GFP, BAG3 and Hsp70 were additionally transfected with FLAG‐p50, as indicated. Diagrams show the percentages of cells with pre‐aggresomal and aggresomal SODG85R‐GFP. Right panel: representative image showing pre‐aggresomal localization of FLAG‐p50 and SODG85R‐GFP. Scale bar, 5 μm.
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Schematic of proposed ncRNA network manipulated by KSHV. circHIPK3 is upregulated by KSHV, partly mediated through ORF57, this leads to increased sponging of miR-30c and increased DLL4 levels. DLL4 has downstream effects on cyclins leading to changes in the cell cycle aiding KSHV replication. Created with BioRender.com.
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(a) Proliferation of cells transfected 3 days earlier with a combination of control siRNA or VHR siRNA together with Mek, Jnk or Mek plus Jnk siRNAs. The data are calculated as percentage of control (control siRNA alone) and represent the mean ± s.d. from three independent experiments. The starting cell numbers were 150,000 or 200,000 and the control cells had increased by 5.3, 5.0 and 13-fold in the three experiments.
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(D) Effect of a p53 mutation on embryonic SA-β-gal activity in the spns1 mutant. The heritable impact of p53 and Spns1 on SA-β-gal induction was tested in each single gene mutant [spns1hi891/hi891 (spns1−/−) or tp53zdf1/zdf1 (p53m/m)] and double mutant spns1hi891/hi891;tp53zdf1/zdf1 (spns1−/−;p53m/m) compared with wild-type (wt) animals at 84 hpf. Scale bar, 250 µm.
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(A and B) THP1-derived macrophages were treated with the TLR ligands Pam3, lipopolysaccharide (LPS), or FSL-1 and analyzed by RNA-seq 8 h later. Circos plots show genome-wide differential expression of protein-coding genes (A) and non-protein-coding genes (B) between untreated and treated macrophages. The outermost to innermost circles show downregulated (blue) or upregulated (red) genes in cells stimulated with FSL-1, LPS, and Pam3, respectively. Chromosomes are indicated by the numbers 1-22 and X and Y.
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immunoblots of the expression of NAD consuming related proteins and their activities in brain tissues from 6-month- old G1 and G3 Tert-/- female mice. The CD38 antibody was validated using the CD38-/- mouse brain tissues.
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(H) Schematic drawing showing the critical biological processes underlying the lifespan extension of age-1; gas-1 mutant nematodes. OXPHOS deficient nematodes display decreased lifespan, diminished ATP production, fragmented mitochondrial network and a phosphoproteome signature associated with enhanced IIS signaling. As a result of IIS inhibition, AMPK and PKA drive a catabolic shift underlying lifespan extension of nematodes carrying OXPHOS defects. Supplementation of xanthine derivatives stimulates survival of mitochondrial mutant animals.
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E Sequential in vitro RNA processing assay using 5'-end radioactively labelled RNA targets corresponding to approximately one repeat of sense and antisense major satellite sequences. The nuage-nuclear fraction from Miwi+/- testes is indicated as N. Total cellular extracts (T) from Miwi+/- testes were subjected to immunodepletion using anti-Dicer antibodies (α-Dicer), with IgG (IgG
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Alterations in the embryonic vasculature in TfebEC-/- mice at E10.5 (mice n=25). Representative images of whole-mount embryos and yolk sacs (i, ii) (scale bars: 0.5 mm). Vessels of the head (iii), ocular (iv) and intrasomitic regions (v) were stained with anti-endomucin Ab (scale bars: 100 µm).
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(B) Circle plots of genes highlighted in (A) (red) and all significantly changing genes (grey).
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MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours A mRNA-levels of different inflammatory cytokines, as indicated. The graphs show relative mRNA-levels, fold increase compared to saline-treated controls, normalized to 18s-content Data information: values are mean±s.e.m. The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant P values: IL6, P=0.0007 (A); TNFα, P<0.0001 (A)
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Confocal micrographs of EphA2 (red) and GPRC5A (green) in TYK-nu and TYK-nu.R treated with 5 µM cisplatin and 10 μM LJH685 as indicated for 72 h. Arrows point co-localization of the receptors. Scale bars: 20 μm.
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(A) Representative images of neurons after 4 days of culture in vitro. GFP expressing plasmid was electroporated into the E13.5 cerebral cortices of WT and KO mice to label neural progenitor cells. After 24 h, the GFP+ cells were isolated and cultured in differentiation medium for 4 days. Scale bar represents 15 μm. (B) Graph shows that the total dendritic length of neurons is decreased when mH2A1.2 is deleted. n = 25 cells from four samples. (C) Statistics show that the number of branch points is reduced upon mH2A1.2 deletion. n = 25 cells from four samples. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**).
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GST pull-down assays. Representative images of n = 3 independent experiments are shown. GST pull-down assay of transiently overexpressed V5-tagged LEMD2 (i), SATB2 (ii), and LEMD2ΔLEM (iii) from HeLa cell lysates using GST, GST-SATB2, and GST-LEMD2(413-503) hybrid proteins.
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(D) Expression of SR-A, FcR, TLR2, TLR4, NRP1 or CXCR2 were measured by flow cytometry analysis.
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(E) Correlation statistics for the localization of 3Flag-MITOL and catalase in the presence of NMS-873. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.
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A. Mapping of enrichment across the positions targeted in lysR. The gray regions in the gene cartoon at the bottom highlights the windows containing targeted residues, with the enrichment map shown above for increasing AEC concentrations. Enrichments are color coded according the legend at the bottom. A histogram plot of enrichment is shown at the right
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d) U2OS mCherry-GFP-LC3 cells were cultured in the presence (+Q) or absence (−Q) of glutamine for 18 h. Red vesicles denote autolysosomes, whereas yellow vesicles represent autophagosomes. Bars indicate numbers of yellow vesicles (autophagosomes) or red vesicles (autolysosomes) per cell±s.d. (e) Images of U2OS mCherry-GFP-LC3 cells cultured for 18 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm.
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HeLa cells were released from single thymidine block, and after 7 hr, BAY 1816032 or DMSO was added for 5 hr. Cells were then fixed and subjected to immunofluorescence staining Example images of anaphase cells are shown (D). Scale bars, 10 μm.
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Kaplan-Meier survival plots for Apcmin/+; Lgr5-creERT2:Ythdf1fl/fl mice (8 mice for each group) treated with or without TAM. Log-rank (Mantel Cox) test. **p<0. 01 (t-test).
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G The pairwise read comparison of the oxidized small RNAs demonstrated an overlap of 10 nt between their 5′ ends.
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(D) Representative pictures of ZEB1 and MITF immunostainings in tumors from patients 2, 3 and 4, before and after vemurafenib treatment. Scale bar = 40 µm. For ZEB1 staining in patient 4, the inset shows a magnification. Arrows point at stromal cells (s). All other cells positive for ZEB1 are tumor cells.
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H, Effect of hlh-30 mutations on the dauer formation phenotype of daf-2(e1370) worms. Data are the mean ± SD of four independent experiments. (***)p<0.001; (****)p<0.0001 by one-way ANOVA with multiple comparison test.
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Steady state levels of endogenous AK2 upon incubation with the DPP9 inhibitor 1G244. As (D) except that cells were treated for 3 or 5 days with inhibitor or DMSO as control. Endogenous AK2 levels were increased upon DPP8/9 inhibition. Reported values are the mean of 5 (3 d) and 6 (5 d) independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05, and *** represents p<0.001.
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D, E IFN-β1 (D) and TRAIL (E) expression detected by FACS in splenocytes is decreased in Smaducin-6-treated CLP mice compared to scrambled peptide-treated CLP mice. Data are representative of three independent experiments.
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Survival curve analysis for hnRNPLL+/+, hnRNPLL+/-and hnRNPLL-/-mice (ten mice in hnRNPLL+/+/+/-group and seven mice in hnRNPLL-/-group).
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(A) Schematic outline of EBNA2, its dimerization domains (END, DIM), the region used by CBF1 to recruit EBNA2 to DNA (WW), the C-terminal transactivation domain (TAD), the nuclear localization signal (N) and the EBNA2 fragments used to map the PLK1 docking site
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C. Lysophosphatidylcholine and lysophosphatidylethanolamine with C16:0 (palmitic acid) and C18:1 (oleic acid).
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Correlation between OCT4-HALO and total OCT4 levels determined by immunofluorescence and HALO labelling. R is Pearson's correlation coefficient.
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Anti-diabetic effect evaluation in ob/ob mice. Mice were injected subcutaneously with vehicle, FGF21 or FGF21SS at indicated doses every day. Body weight (B) was measured Error bars show the SEM of ten independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student's t-test) vs vehicle.
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(A) Flag‐SUMO2 was expressed in HeLa cells by transient transfection. Flag‐SUMO2‐expressing cells were visualized by indirect immunofluorescence. The localization of endogenous PELP1 was monitored in Flag‐SUMO2‐positive cells (green arrowheads) or untransfected cells (white arrowheads) using anti‐PELP1 antibody.
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(B) Correlation between CSF p-tau235 and p-tau231 in the whole ALFA+ cohort (Spearman's rank correlation: rS = 0.80, P˂0.0001). Assay cut-offs were determined as the mean + 2 SD of the A-T- group (defining p-tau231 and p-tau235 positivity or negativity in each participant). Cut-off values are indicated in red (19.92 and 9.59 pg/mL for CSF p-tau235 and p-tau231 respectively) and displayed with black dashed lines, resulting in four quadrants, each of them representing the four different positive or negatively status for each biomarker. Discordant cases with the sequential phosphorylation hypothesis ([231-/+235]) showed on the lower right quadrant (2.35% of the total, 9 participants out 383). All samples were run in singlicates.
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(B) primary human bronchial epithelial cells were infected with one of several P. aeruginosa mutants. Cell lysates were evaluated by immunoblotting.
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(A) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3). Cytoplasmic and nuclear extracts were isolated at the indicated time points post infection, and levels of ICP27, RCC1, and GAPDH were determined by Western blotting.
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Abundance distribution of all transcripts detected in all tissues (grey); the fraction of detected proteins is shown in blue and the fraction of transcripts for which no protein was detected is shown in orange.
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Lost preference for the novel object (nov) comparing to the familiar object (fam) in AP-2µ KO mice tested 24h after training in NOR (WTfam: 27.51±6.77, WTnov: 72.49±6.77, pWTfam vs WTnov=0.000; KOfam: 43.9±13.12, KOnov: 59.91±13.12, pKOfam vs KOnov=0.469, N=7 WT and 8 KO mice). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant.* indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.
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(G) Representative pictures of SA-β-galactosidase staining in control (passage 15) and P1's primary fibroblasts (passage 6). Results are expressed as the percentage of SA-β-galactosidase-positive cells (averages, lower panel). Control: n = 436; P1: n = 436. A test to compare two population proportions was applied.
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C) Quantification of Myc-CENP-A assembly in M18BP1-depleted extract complemented with the indicated M18BP1-1 species. Graph shows mean immunofluorescence intensity at centromeres normalized to reactions complemented with WT M18BP1-1. Error bars show SEM of at least three independent experiments.
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