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(D) Cells were transfected by siRNa as indicated followed by incubation in serum-starvation media for 24 h, and immunostained for EHD1 (red) and γ-tubulin (green). Scale bar, 10 μm. (E) Quantification of the percentage of cells with EHD1 in cilium or in the distal end of basal body as shown in (D). Data represent mean ± SD (n=3 experiments), 150 cells were scored per condition per experiment, *P < 0.05, Student's t-test.
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CY and CpdA, but not bendamustine or CpdB, inhibited HDAC activities in cancer cells. H1650 cells were treated with 20 μM CY, 20 μM CpdA, 200 μM CpdB, 200 μM Bend, or 10 μM SAHA for 12 h, and cell lysates were blotted for histone acetylation markers. Actin was used as a loading control.
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B) Cytosolic ATP production rates. Each data point represents the mean ATP production rate of one well (~12 technical replicates per measurement, N=4 independent assays.).
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C-E. Relationships between pairs of phenotypic traits. C. Length of the elongation zone LEZ versus its number of cells, NEZ. Panels from left to right: the Ruler, Timer and Sizer models, and epidermal Col-0 data. Symbols in the three left-most panels represent simulated data of individual simulated root files (gray triangles for Ruler, pink squares for Timer and red diamonds for Sizer, n=122 each). Data from individual epidermal files in Col-0 is in green circles (n=122 from day 6, 8 and 10 post germination (Tables EV1, EV2)). Dashed lines are minimum square linear fits. Pearson correlation coefficient (r) for each pair of data are indicated. p stands for the p-value using standard Pearson correlation test. The epidermis exhibits of the number of cells in the EZ with its length, and also with 1/ln(rEZ). The epidermal mature cell length does not have correlation with the inverse of the elongation factor. These features are only reproduced in the Sizer model. For each model, the simulated roots and cells differ in the threshold value for cell elongation termination, the cell elongation rate, the meristematic activity and the initial cell length, according to a variability inferred from the epidermal Col-0 data (Appendix Fig. S4B-F). The parameter values are the same for the three models except for the threshold for cell elongation termination, which is specific of each model and has relative variability of 35% (Ruler), 7% (Timer) and 26% (Sizer) (Table EV3). Parameter values selected such that no statistical significance is found between model (for each model) and epidermal data in any of five phenotypic traits (Wilcoxon-rank sum test, p-value >0.01, Table EV4, Appendix Fig. S4B-F). Continuous lines are theoretical predictions for each model (Appendix Text S1E). All models have the same dependence on the threshold value for differentiation (blue line) but each model has its own dependence on the the spatial profile of cells along the EZ (rEZ) (lines in gray for Ruler, black and red for Timer and red for Sizer). The Timer model depends on the cell elongation dynamics and, in contrast with the Ruler and Sizer models, its relationships are not univocally defined by the spatial profile of cells along the EZ (rEZ). For the Timer model, the continuous lines represent the theoretical prediction obtained when either relong (black line) or Rprod (red line) changes. For all the boxplots, boxes represent the interquartile range (25th-75th percentiles, with the median indicated by the black horizontal line) of the distribution, whiskers extend to the minimum and maximal values excluding outliers. Outliers are those values more (less) than 3/2 of upper (lower) quartile.
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In WT neurons, AP-2μ is required for transport of BACE1- containing autophagosomes and late endosomes en route to lysosomes. Absence of AP-2μ causes defective axonal trafficking of BACE1 leading, which results in increased amyloidogenic processing of APP.
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A We co-transplanted barcoded wildtype (WT) HSCs and B cell deficient (uMT-/-) competitor HSCs into irradiated recipient mice. WT HSCs were used as competitor HSCs in the control group. 7 months after the primary transplantation, we harvested peripheral blood cells and purified both WT HSCs and competitor HSCs from the bone marrow. HSCs from one primary recipient mouse were transplanted into one secondary recipient mouse. 4 months after the secondary transplantation, peripheral blood cells were sorted into granulocytes (Gr), B, CD4 T, and CD8 T cells for population and clonal level analyses
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(I) IHCs of KIRC clinical samples were performed using antibodies against GDH1 or RNF213 (upper panel). The correlated IHC signal between GDH1 and RNF213 in KIRC samples was calculated (lower panel). The cor.test was used to determine the p-value. Scale bars: 50 μm.
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D Hanging test performance was assessed in ERp57WT (n=41), ERp57Nes+/- (n=32) and ERp57Nes-/- (n=12) mice.
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B The ubiquitination of ENTREP. The immunoprecipitated samples using anti-FLAG antibody under the denaturing condition were separated by the SDS-PAGE. The gel area corresponding to band #A, #B and #C of ENTREP-FLAG were applied to the ubiquitin-AQUA/PRM analysis. The absolute number of each ubiquitins of ENTREP #A, #B and #C were calculated by subtraction of those of the corresponding area of the control vector sample (Appendix Table S2 for raw data of the ubiquitin-AQUA/PRM analysis). Based on the ratios of each ubiquitins to the total ubiquitin of indicated gel area, which are showed as Circular graphs (ENTREP #A, #B and #C), the number of ENTREP-linked ubiquitin (Ub) is summerized as schema: #A is modified with one Ub, and about two thirds of #B molecules and half of #C molecules are modified with two and three of single Ubs (multi-monoubiquitin), respectively. The result was based on three biological replicates.
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CIP2A expression correlates with mTORC1 activity in primary human breast tumors. TMAs including 33 normal breast tissues and 210 primary breast carcinoma samples were analyzed by semiquantitative immunohistochemistry for the expression of the indicated proteins and mean scores (arbitrary units [a.u.]) for duplicate samples were averaged and plotted according to mean CIP2A expression (left). The red lines are the regression lines, and the stippled red lines define the 95% confidence intervals. Statistical analysis of data was performed using NCSS 2007 and Prism version 5. Examples of the staining of breast cancer (a) and normal (b) cores indicated in the P-T389-S6K1/CIP2A plot are shown on the right. rsp, Spearman's correlation coefficient.
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A. Western blot showing equal BAZ2A levels in wt-ESC and F/H-BAZ2A ESC line containing FLAG-HA sequences at the N-terminus of both Baz2a alleles. Whole cell lysates from equivalent amounts of cells were analysed. SNF2H, and Tubulin are shown as protein loading controls.
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(H) A schematic diagram depicts critical domains of PAQR3 involved in the interaction with ATG14L and Beclin1 respectively.
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(A) WT cells expressing genomic Atg36-PtA and Pex11-GFP on a plasmid were grown in glucose for 22 h, transferred to oleate medium, and then shifted to SD‐N medium. Samples were taken at the times indicated and processed for western blot with peroxidase anti‐peroxidase complex to detect Atg36-PtA (top panel) or anti‐GFP to detect Pex11-GFP pexophagy (bottom panel). GFP* indicates the relative protease‐resistant degradation product and reflects vacuolar breakdown.
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(A) A group of exclusively ATM-dependent sites that were ATM-dependent one hr after NCS addition, were still phosphorylated or dephosphorylated 4 hr after treatment, but were not affected by ATMi at that time point. Box plots depict 18 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).
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Nucleosomal array oligomers are globular.A Nucleosomal array oligomers were stained with DAPI and examined using FM (fluorescence microscopy) as described in the Materials and Methods section. Shown are representative images obtained in 4.5 mM and 10mM MgCl2.B Control FM images obtained in 0, 1 and 2.5 mM MgCl2.
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MoLC from six different donors were transfected with siCTR or siS100A9 and challenged with HIV-GFP vectors for 48h. S100A9 expression (left graph) and the percentage of GFP+ cells (right graph) were analysed by RT-qPCR and flow cytometry respectively. Flow cytometry data are presented as fold relative to siCTR-MoLC. Data represent means of fold values for each read-out from six donors (n=6).
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(C) Uptake characteristics of T-LipoAS by primary human hepatocytes demonstrate competitive inhibition by cyclosporin A- and energy dependence, confirming active uptake by organic anion transporters (OATPs). The data points are depicted mean ± SD from two batches of primary human hepatocytes in quadruples.
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(d) Reduced lifespan of HdhHD/+ Dnchc1Loa/+ mice. HdhHD/+ Dnchc1Loa/+ (red line) n = 13, HdhHD/+ Dnchc1+/+ (green line) n = 9, P = 0.038. Mice were subjected to humane end points, and so the survival curve reflects the onset of the end points rather than the complete lifespan. Asterisks (*) represent mice that were found dead without reaching humane end points. No mortality of Hdh+/+ Dnchc1Loa/+ mice was observed.
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(B) Comparison of emission spectra of various IAEDANS-labeled Dyn1 RCL variants (D352C, Y354C, S357C, I365C, H367C), upon Trp excitation at 295 nm.
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E Population composition in male HD bulk edited, sorted NGFR+ or sorted NGFR- CD4+ T cells from (B) (n=7 for each group). Friedman test (to account for the same donor) with p-values adjusted with Bonferroni's correction to account for multiple testing. Mean ± SEM.
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D. Heat map showing accessibility signal (log1p of fold over background) at p300-positive ATAC-seq peaks surrounding pluripotency genes (ventx1/ventx2, pou5f3 and sox2). The row labeled 'random' shows accessibility signals at random genomic loci.
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J. Example stills from a spinning-disk time-lapse recording of a neurite transfected with mRFP and GFP-MT+TIP. Red line denotes the location of LS. Scale bar = 5 µm. K. Kymographs and schematic representations of time-lapse recordings of the distal axon as shown in (J) following LS, for different time points (day 7; day 13) and conditions (control; Centrinone-B). Red line and red arrowhead denote location and time of LS. Scale bar = 5 µm. L
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D. Residual human monoclonal antibodies in the serum of representative surviving mice at 100 dpi (LOD: limit of detection = 0.02 µg/mL).
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Immunofluorescence showing Olfm4+ve cells in Lgr5-DTReGFP duodenums. Cell membranes are shown with b-catenin and nuclei were counterstained with DAPI. Quantification of Olfm4+ve cells per intervilli regions in duodenum and ileum. Each symbol indicates the value for a given embryo.
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C qRT-PCR-validation of HSATIII knockdown. The graph shows the qRT-PCR level of HSATIII RNAs in control and HSATIII knockdown cells under three conditions: 37°C, 42°C for 2 h, and thermal stress followed by recovery at 37°C for 1 h (Recovery). Expression levels were calculated as ratios to GAPDH mRNA and were normalized to the levels in control cells under thermal stress conditions (42°C for 2 h). Data are shown as the mean±SD (n=3). HSATIII RNAs ratios (%) are indicated.
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(A-D) Expression of LGG-1 in wild-type embryos at the ∼100-cell (A and B), the ∼200-cell (C), and the fourfold (D) stages. (A) DAPI image of the embryo shown in B.
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Fluorescence immunohistochemistry of the ependymal surface of the lateral ventricle of the mouse brain. Cilia are visualized using an antibody for acetylated alpha tubulin (green). Cwh43 is visualized using a specific anti-Cwh43 antibody (red). Nuclei are counterstained using DAPI (blue). Arrowheads point to motile cilia and scale bar is approximately 5 µm.
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(A) MYC2, PDF1.2A and NIMIN1 expression in ANAC032 transgenics compared to WT after spraying with Pst DC3000 (24 hpi) normalised to their respective controls. FCh, fold change. Means ± SD (n = 3; 'n' represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots).
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immunofluorescence staining of desmin (green) (G) in GA muscle from WT and mGPDH-/- mice at day 7 post CTX injection
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Cleavage of full length TNF/Eiger by ADAM17 leads to the production of soluble TNF/Eiger, which can engage the TNF receptor, Grindelwald. TNF signalling inhibits the production of mitochondrial ROS inside pigmented glial cells, through unknown mechanisms. In the absence of TNF signalling, glial generated ROS combines with ROS from neighbouring photoreceptor neurons to trigger production of LDs. Upon their later dispersal, the combination of fatty acids and ROS leads to high levels of toxic peroxidated lipids, which are toxic to the glia. Finally, glial degeneration is followed by neuronal degeneration.
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Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs (n=3 independent experiments; 46-54 cells).
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C 200 pmol of indicated oligos treated as in (B) were loaded on a native polyacrylamide gel with the selective G-quadruplex staining N-methyl mesoporphyrin IX. Asterisk (*) indicates the second NMM-positive band obtained upon SMaRT and γ-oligo interaction. Data information: Representative gels from at least three independent experiments are shown.
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Our current model predicts that defects in pantothenate kinase 2 impair the phosphopantetheinylation of mtACP and lead to impaired complex I activity (Fig. 2E), iron-sulfur biogenesis (Fig. 2C), and mitochondrial fatty acid synthesis. Lipoic acid modification is required at the E2 domain of PDH complex. Decreased production of lipoic acid in mitochondria would diminish activity of this enzyme (Fig. 2D). The clinical features of PKAN, including abnormal iron accumulation and parkinsonism, can be explained by these metabolic defects. Their rescue by exogenous 4'-phosphopantetheine suggests that the CoA pool critical for mtACP phosphopantetheinylation is at least partially dependent on pantothenate kinase 2.
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G. Caspase 3/7 activity in H295R/TR N-FlagFATE1 cells cultured in basal conditions (white histograms) or in the presence of Dox (red histograms) and treated with mitotane (50 μM) (mean ± SEM; n=5-8 with 3 replicates/condition). **p<0.01.
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lacZ mRNA resulting from the different degrees of induction as well as aceB mRNA in the same cultures were measured by qPCR and plotted as fold-change increase compared to that in the absence of induction. shown both as linear (left plot) and log2 (right plot) scales. The dashed line in the left plot shows a linear fit. aceB mRNA abundance is inversely related to the lag-time (D). Means of N=3 and N=5 biological replicates are shown for lag-times and expression levels respectively in panels B-D. Error bars denote SD.
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U87 cells were treated with increasing concentrations of LXR623 in the presence or absence of exogenous ATP for 7h. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4.
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C Immunostaining of tumor and LN from patient ER-0064 for expression of ER, PR and pan-cytokeratin. Insets show sections stained with control isotype antibodies. PR, progesterone receptor. Scale bar, 100 μm.
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GAS-luciferase reporter assay in Vero cells infected with BTV-8 or mock-infected (Control) and treated wit 5 ng/ml of IFN-γ. Luminescence fold induction was measured at different time points p.i. Mean luciferase activity fold inductions in control or BTV-infected in three experiments are presented
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(c) A representative example for enrichment of CLV3 transcript in mCherry-positive nuclei determined by qRT-PCR and normalized to wt (N = 1).
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Co-IP assay was performed to detect the interaction between CULLIN3 with SPOP WT or Q165P (G).
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(B) Overlap of phosphoproteins that were "down-regulated" by nitrate at 5 min (blue) and 20 min (light blue).
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(A) Time course experiment of HuR binding of miR-122 replaced from Ago2miRNPs. A schematic representation of in vitro miRNA binding assay of HuR (left panel). Equal amounts of recombinant HuR (rHuR), after indicated time of the miRNP interaction on FLAG-beads, were immunoprecipitated and HuR associated miR-122 levels were detected by qRT-PCR (right top panel). Ago2western blot detects its presence in the FLAG beads used in the assay and its absence in HuRimmunoprecipitated materials. HuRwestern blot was used to confirm its presence and quantification of its levels in the immunoprecipitated materials, at different time points (right bottom panel).
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Loss of cyclin F induced E2F7/8-dependent Homologous Recombination deficiency. HeLa cells that were stably transformed with pDR-GFP were transfected with siRNA as indicated.48h after the initial transfection, cells were harvested for Flow-cytometry. GFP positive cells were gated (Fig EV5A). Relative HR efficiency were adjusted to the scramble siRNA condition. Data represent averages ± SEM (n=3); **P<0.01 (Student's t-test). n.s.: not significant.
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Three-day-old seedlings of Col-0, MPK3SR, MPK3SRarr1/12, and arr1/12 were transferred to 0.5 μM NA-PP1-containing 1/2 MS medium for 2 d to inhibit MPK3TG in MPK3SR and MPK3SRarr1/12 and then the plants were transferred to 0.2 μM NA-PP1-containing 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. Pictures were taken and the elongated root length was determined 3 d later (A-B). Changes in survival rates (C) and fresh weights (D) were examined 10 d later. The fresh weight of NaCl treated seedlings/fresh weight of NaCl untreated seedlings ratios were labelled in the columns in (D). Scale Bar, 1 cm.
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(B) GCSF has limited effect on mediating transcription of RA-target genes that promote granulocytic differentiation. Dot line, non-direct effect on gene transcription; white arrow, limited effect on gene transcription.
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(D) HeLa CCL2 cells co-transfected with HA-Ect2 and GFP-β-catenin were synchronized using a double thymidine block and released using medium containing 200 ng/mL nocodazole (Noc.) at the indicated time point. Cells at each release time point were harvested and used for immunoblot analysis using anti-HA antibodies. Immunoprecipitated beads were analyzed by SDS-PAGE followed by immunoblot analysis. Band intensities were quantified using the ImageJ program and normalized to the level of immunoprecipitated HA-Ect2 protein, and the relative values of band intensities are marked.
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D. Close up view on the relative spatial orientation of the conserved basic patch in Elp2 and the potential interaction site between Elp456 and a region of Elp2 important for tRNA modification activity.
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(a,b) Fluorescent microscopy of (a) NDP52 or (b) P62 in cells expressing LINE-1-MS2 and MS2-GFP. (c,d) Fluorescent microscopy of endogenous LINE-1 RNA and (c) NDP52 or (d) P62. (e) Four-colour confocal microscopy of NDP52, P62, GFP-DCP1A and mCherry-TIA-1. (f) Percent of foci of NDP52, P62 or neither co-localizing with GFP-DCP1A or mCherry-TIA-1. *P=0.007 GFP-DCP1A, P=0.00005 mCherry-TIA-1, analysis of variance (ANOVA). (g) Percent of foci of NDP52, P62 or both co-localizing with GFP-DCP1A or mCherry-TIA-1, n=674 stress granules, n=53 P-bodies. *P=0.043 GFP-DCP1A, P=0.028 mCherry-TIA-1, ANOVA.
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Distribution of the positions of the AP-1 motif relative to the summit of the TSS-distal, CFPAC1-specific FOXA2 peaks. The graph indicates the average density in 20 bp bins in a window of 500 bp.
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(D) Effect of Parkin silencing on depletion of mitochondrial DNA caused by HCV infection. Huh7 cells transfected with NT or Parkin-specific siRNA pools were infected with HCVcc. At day 3 post-infection, mitochondrial DNA levels of ND-2 and COX-2 were analyzed by real-time qPCR. GAPDH was used to normalize changes in ND-2 and COX-2 gene expression (mean ± SD; n = 3; *p<0.01). (A, B, and D) P values were calculated by using an unpaired Student's t-test.
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E. LRRC23 localized to the nucleoli in U-2 OS cells (HPA057533). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.
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(A-C) Transmission electron microscopy of β-cells from Abca12tm1d and control mice at 8 weeks of age. Panels A'-C' depict higher resolution images from the boxed regions in A-C (scale bar = 1μm).
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Astrocytes were selectively isolated by MACS from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at days 0 and 15 p.i., respectively. Relative expression of STAT1 mRNA was determined by quantitative real-time PCR. Data show the relative increase of STAT1 mRNA over that of unimmunized control mice (n = 3 for all groups) (mean + SEM).
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A Representative IF staining for costaining with TET3 and Mash1 antibodies in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both TET3 and Mash1. A' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). A: Scale bars, 20 μm; A': Scale bars, 2 μm. B Quantification of the number of costained TET3+ and Mash1+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; ***p<0.001; Student's t-test).
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D Western blot analysis of Bax, BaxTBak, Bak and BakTBax localization expressed in HCT116 Bax/Bak DKO cells. Cytosol (C) and heavy membrane fraction (HM) of HCT116 Bax/Bak DKO cells are displayed. GAPDH and VDAC serve as fractionation controls. n = 3.
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Wnt inhibition reduces the expression of HR and FA pathway genes in APC-mutant COLO320HSR cells. COLO320HSR cells were seeded in 6 well plates, treated with control siRNA, siβ-catenin, DMSO or G007-LK for 72 hours and then the expression of the indicated genes was measured by qRT-PCR. The horizontal lines represent mean of replicates
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(F) Correlation between N-specific T cell activity and intracellular neutralization activity. Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical analysis was performed using a semilog non-linear fit
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C. Soluble chromatin extracts were prepared from preadipocyte or induced 3T3-L1 cells stably expressing control (EV) and Flag-WDTC1 proteins. H2A monoubiquitylation was detected by H2AK119ub1-specific antibody and total H2A served as loading control. Relative intensity represents ratio of H2AK119ub1 over total H2A signal normalized to EV control.
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B. Expression comparison of the SATS isoforms (SATS), common isoforms (common) and Refseq genes (Refseq) in mESCs. The Y-axis represents the log2 transformed average FPKM values of each isoform or gene in three mESC lines (F145, F146 and F147). The FPKM values in each cell line was calculated by the reads mapped to the first exon and normalized by the exon length. Red dots represent the mean expression level of isoforms or gene in each group. The SATS and common isoforms from the same gene are connected by grey lines.
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D Representative microscopy images showing Fab-PLA results measuring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. PLA signals are shown as red dots and the nuclei are visualized by DAPI staining. Scale bar: 5 μm.E The Fab-PLA results are quantified by Blobfinder software and presented as box plots. The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. Data from treated samples were compared to data from resting cells, p-values were calculated by Kruskal-Wallis one-way analysis of variance (ANOVA).
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Testicular maturation arrest in the patient (WHT3759). Testicular tissue obtained by biopsy was sectioned and stained with hematoxylin/eosin. Seminiferous tubules contained meiotic germ cells such as zygotene (ZS, arrowheads) and pachytene spermatocytes (PS, arrows), but no post-meiotic germ cells such as round spermatids were detectable. Scale bar, 25 μm.
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Forebrain slices from WT mice were treated with vehicle (water; lanes 1,5; numbers on bottom) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(D) α11.2 and GluA1 were concurrently IPed from same samples as in (A) by simultaneous addition of anti-α11.2 and -GluA1 antibodies before probing and stripping/re-probing upper part of IB for pS1928, pS1700, and total α11.2 (top three panels) and middle part for pS845, pS831, and total GluA1 (bottom three panels).(E-H) For quantification, pS1928 and pS1700 IB signals were normalized to total α11.2 and pS845 and pS831 signals to total GluA1 (**p<0.01, ANOVA).ISO-induced displacement of the β2AR from α11.2 (see A) rendered α11.2 (but not GluA1) refractory to re-phosphorylation of S1928 and S1700 upon a second ISO application of α11.2 (D, compare lanes 5 and 6).
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Representative Western Blot (D) and the relative bar plot (E) showing collagen expression in Fam134b knockout MEFs overexpressing wild type and the ∆LIR Fam134a protein.
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A. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 30 min or were untreated. The cells were washed with PBS and the media was replaced with fresh media containing staurosporine (500 nM). Whole-cell lysates were harvested and analyzed by the Western method;
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Human induced pluripotent stem cells (iPSCs) derived from a FXS patient (FXS-iPSC) and the isogenic rescue cells (C1_2-iPSC) were analysed by immunofluorescence microscopy Examples of co-localization events of re-expressed FMRP and Nup133 are shown in (G).
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(g,h) HA-IP pull-down products from HA-HACE1MEF (HA-HACE1) and Hace1 KO MSCVMEF (MSCV) immortalized in HA beads were further subjected to Co-Co-IP with WT mouseheart lysates, and the pull-down products were analysed by western blot using the indicated antibodies. HACE1 and those proteins with which it interacts were still detectable (g). There were also increased ubiquitinated proteins in the Co-Co-IPheart tissues (h).
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(b) Bax/Bak−/− MEFs were transiently transfected with 10 μg of siRNA for Bcl-2, Bcl-x, or Bax (negative control) for 24 h. Expression levels of Bcl-2, Bcl-xL and Lamin B (loading control) were analysed by Western blot analysis.
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HT29 cell survival 18h after treatment with TNF+BV-6+z-VAD-fmk to induce necroptosis. Cells expressed lentivirus constructs encoding 1-90 or 1-277 fragments of M45 and were cultured +/- the addition of coumermycin A1 to induce dimerisation. N=2 for M45-90 gyrase +/- coumermycin A1 and 4 for M45-277 gyrase and for empty vector, +/- coumermycin A1
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K Violin plot showing the percentage of KI67 and hECAD double+ cells of total hECAD+ HBECs derived from 11 different patients upon 21 days of CTRL (n=360), P4 (n=180), DSG (n=147), GSN (n=298), LNG (n=284), CMA (n=115), CPA (n=156), DSP (n=123) treatment. Red lines show medians. L Violin plot showing percentage of AR+ intraductally-engrafted HBECs derived from 11 different patients upon 21 days of CTRL (n=258), P4 (n=97), DSG (n=173), GSN (n=159), LNG (n=168), CMA (n=68), CPA (n=76), DSP (n=135) pellets. Statistical significance was assessed by fitting a generalized linear mixed model with gamma distributions, with batches and patients as random variables and CTRL as reference, median in red. J-L dashed line shows 0%.
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Activity assay of mGPDH in GA muscle from C57BL/6J mice at day 0 and 7 after CTX injection
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Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection on the splenic lymphoma burden of RC-K8-transplanted MISTRG6 mice (H), all assessed at around four weeks post tumor cell injection.
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(C) FYCO1 can self-interact in a CC-dependent manner. Full-length GFP-FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of myc-tagged FYCO1 in rabbit reticulocyte lysate. S35-labeled FYCO1 complexes were immunoprecipitated with anti-myc antibody, separated by SDS-PAGE, and visualized by autoradiography. IP, immunoprecipitation.
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(E) Patient samples from METABRIC dataset classified as TNBCs (N=299) were assigned to the groups "PTEN-low" when falling in the lower quartile for PTEN expression, "PTEN-mut" when harbouring a non-synonymous mutation on PTEN gene, "PTEN-WT" in all other cases. Comparison of the expression of GNB2 between the PTEN low or mut and the PTEN-WT groups. Box & Whisker plot (Median/IQR/1.5*IQR Whiskers). P value calculated by unpaired t-test.
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(H) GST pull-down showing the interaction of purified GST-CP110-N with purified His-ENKD1-M.
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(A) Ratio of phosphorylated eIF2α (p-eIF2α) over eIF2α in MM cells exposed to 800 nM BoxA or increasing concentrations of CXCL12 for 24 hours (n=2). β-actin is shown as loading control. Data information: bars and error bars represent mean ± SD; statistics: one-way Anova plus Dunnett's post-test. In all panels, *p<0.05, **p<0.01, ****p<0.0001.
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c) Pull-down assay to assess the competition between Beclin 1-Atg14L and Beclin 1-UVRAG complexes. Increasing amount of His6-tagged Beclin 1 CC domain was added to a mixture of Atg14L and UVRAG proteins corresponding to their respective CC region. The Beclin 1-Atg14L/UVRAG complexes were pulled down by Ni2+- nitrilotriacetic acid (NTA) agarose beads and checked by SDS gel.
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Representative images of the distribution of Fib-Tau (red) 8 h and 24 h after injection. Dendrites are immunolabeled using MAP2 antibody (green). 8 h after injection, both diffused and clustered distribution of exogenous Fib-Tau is observed in stratum oriens and CA1 cell body region No exogenous Fib-Tau is detected in the adjoining cortex, stratum radiatum and entrorhinal cortex 24 h after injection, clustered distribution is primarily restricted to the pyramidal (CA) cell body layer (D). Uptake of Fib-Tau is seen in some cells in both stratum oriens and pyramidal cell body layer (D). Paired-t-test performed between '8/24h-right' and '8/24h-left'; Unpaired t-test performed between '8h-left' and '24h-left' columns (n is number of animals: 6 for 8h and 5 for 24h); ***p<0.001, **p<0.01, *p<0.05, ns= not significant. Note: The images presented in panels C and D were independently acquired images under non-identical exposure setting at low magnification for display purpose Data information: Scale bar, 10µm
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The notum bristle phenotype caused by HP1c depletion can be rescued by human homolog HP1γ OE, as indicated by the enlarged views in the left bottom panes. Scale bars, 100 μm.
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RT-qPCR analysis confirmed the overexpression of HES1 mRNA after DOX induction. The ACTB gene was used as a control. The data was normalized to the mRNA level in empty vector control cells.
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(E) Hypothetical model of membrane targeting of Atg16L1. Interaction of Atg16L1 with FIP200 leads to proper targeting of this complex to the autophagosome formation site (middle). Without FIP200, puncta formation of Atg16L1 is impaired probably owing to a self‐inhibitory role of the C‐terminal WD‐repeat domain (left). If the WD‐repeat domain is deleted, the N‐terminal half of Atg16L1 localizes to aberrant membranes as well as the autophagosome formation site (right). KO, knockout; MEFs, mouse embryonic fibroblasts.
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C-D, cell viability of residual MDR cells (RMCs) and MDR cells (MCs) of (C) Du145TXR or (D) MCF-7ADR cells using the same combination treatment for 24 hours on the indicated days. Student's t test was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments.
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K Percentage of expanding clones in the B cell lineage from the primary recipients that continued to expand in the B cell lineage of the secondary recipients. Expanding clones are defined as those producing more than 0.1% of WBCs. Data information: Data were collected at month 4 after secondary transplantation and presented as mean ± SEM. n=7 mice for the primary control (WT+WT) and deficient co-transplantation (WT + uMT-/-) groups. n=4 mice for the secondary transplantation control group (WT + WT) and n=6 for the secondary deficient co-transplantation group (WT + uMT-/-). *p<0.05 and **p< 0.01 (two-tailed and two-sample equal variance Student"s t-test)
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C. Most enriched functional annotations in genes differentially expressed in Kdm6apKO.
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G) Representative confocal microscopy images of U2OS BAX/BAK DKO cells of the interactions between RA-BAX and GB-DRP1 variants, shown as green signal, 3 h after apoptosis induction. Mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm.
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B. RNA expressional changes of FoxM1, Id1, and Jnk3 after adeno-Ctrl or adeno-FIJs treatment after MI.
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A Chitin-induced callose deposition in the mapkkk5 mutants. Seedlings were analyzed at 18 hours after treatment with 10 μM chitin. Representative pictures from the biological replicates are presented. Data are means ±SD from three independent biological replicates, where each biological replicate consists of two technical replicates. The asterisks indicate statistically significant differences from the WT controls by Student's t-test (P < 0.05).
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qRT-PCR detecting mRNA transcript level of ten selected highly expressed genes The numbers indicate the gene ID.
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(A) Overview of a reconstructed superresolution image of GFP-Bax overexpressing HCT116Bax/Bak -/- cells stained with AF647-anti-GFP-nanobodies Image was acquired on fixed cells 7 h after apoptosis induction with STS. Dotted line shows the cell shape (see Fig EV3). Scale bar, 5 µm.(B) Gallery of typical GFP-Bax WT non-random structures during apoptosis on HCT116Bax/Bak -/- cells. Scale bars, 100 nm.
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G Mean mitochondrial Ca2+ peaks response to 100 μM CCh. Mean ± s.d., student t-test, ***p ≤ 0.001, n = 3. Data information: Error bars for some points are too small to be visible.
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Anti-VEGF/R treatments exacerbate the invasive capacity quantified as capsular invasion in the tumor kidney interface in some Ren-PDOXs. Moreover, the evaluation of lung metastasis confirms the pro-malignant effects of antiangiogenics in some Ren-PDOXs, recapitulating the inter-patient variable response.
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A. Individual traces of mEPSCs of both control and naspm treated neurons after TTX, TTX + DHPG, and TTX + DHPG + 2APB/dan treatment.
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C, D. Levels of lyso-Gb3 were measured by LC/MS in plasma and extracts of tissues of mice engrafted with HDo-derived (C) or FDo-derived (D) TRaMs.
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(G) Nonlinear single-exponential regression was used to fit the average of radial RBP-3 τdonor line scans in control oocyte with the one-phase decay model.
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C Three representative NFR-resistant HeLa clones were analyzed by Immunoblot for eEF2K expression level and eEF2 phosphorylation upon NFR and compared to parental HeLa cells. For treatments, medium was replaced for 6h with medium containing DMSO (Mock), increasing doses of NFR or 10 μg/ml tunicamycin (TM), or for 1h with PBS for starvation (Starv.). Tubulin is used as loading control.
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D) Average +/- s.d. (dot) host cell numbers per well for three independent biological replicates under the different AIP concentration.
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(A) Western blot analysis of the CTH level in PC3-T2 cells with CTH overexpression by pCMV-CTH-HA or PC3-B2 cells with CTH knockdown by siCTH-1.
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A, Schematic outline of plasma membrane D4H labeling. CM and LPDS indicate complete medium (10 % FBS in DMEM) and 5 % LPDS in DMEM, respectively.
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B Endogenous linear Ub chains in total cell lysate (TCL) of S2 cells expressing human Flag-OTULIN WT or catalytically inactive C129A mutant. TCLs enriched with GST-Linear-TUBE were examined by immunoblotting using anti-linear Ub antibody. Expression of OTULIN was analyzed by anti-Flag antibody and loading of TCL was detected by anti-Tubulin antibody. Input of GST proteins was analyzed by Ponceau S staining. * : nonspecific band.
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To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with carbonyl cyanide m-chlorophenyl hydrazone (10 μM CCCP) (B) Protein complexes were separated on BN-PAGE and visualized by Coomassie staining and autoradiography. 1 hour after the stimulation, significant increase in 35S-labelled proteins was observed (B, n=4 biological replicates, * , ** p=0.0023; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S-methionine into respiratory chain complexes when samples were incubated in the presence of CCCP (B; n=4 biological replicates, * p=0.02, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test)
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E. HEK293 cells overexpressing the wild type form of GSNOR (GSNORwt) or an empty vector (Empty) were transfected with siRNA against Pakin (siParkin) or control siRNA (scramble, siScr). Afterwards, they were subjected to combined treatment (200 μM H2O2 + 400 μM DPTA) and viability assessed by Alamar blue (AB) fluorescent assay. Data, shown as fold change of viable cells, refer to AB fluorescence (relative to untreated cells, arbitrarily set to 1) and represent the means ± SD of n = 6 independent experiments. **, p<0.01; n.s., not significant.
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