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SAMHD1 knockdown enhances EV71 replication. The stable cell lines pLKO.1 and sh-SAMHD1 constructed in RD (H) cells were infected with EV71 at a MOI of 0.1 and 0.05 respectively, and cells and supernatants were harvested at the indicated time points. IB analysis of EV71 VP1 and SAMHD1 in cells was performed with tubulin as a loading control. EV71-VP1 protein in the supernatants was detected after ultracentrifugation.
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SUMO conjugation assays performed under multiple turnover conditions using Siz2∆CT and different RPA-DNA complexes. SDS-PAGE gels were stained with Coomassie. Bands for Mw marker are fully annotated in Figure 1B.
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B Western blot analysis of PIWI proteins in the locust brain. Tubulin was used as an endogenous control (n=2 biological replicates). The bars represent the mean of band intensity
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(C) Administration of calcium chelator BAPTA prevents ER stress-induced apoptosis in EI24 knockout cells. Cells were cultured in the presence of 50 μM BAPTA for 1 hr and then treated with or without 4 μM TM for 8 hr. Percentages of early apoptotic cells were measured by flow cytometry. Data information: N ≈ 10000 cells for one biological replicate. Data are mean ± SEM (n = 3 or 4 biologically independent replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test
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M Hematoxylin and eosin staining of liver tissue the same animals as in (C) after 24 h of Tm injection (magnification × 400). Immune cell infiltration was indicated with a black arrow. Scale bar = 50 μm. Representative images were shown from five animals per group analyzed.
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G Immunoblot analysis of LM and HR1 cell lines showing NFIB and FOXP1 protein levels. ERK2 served as a loading control.
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(A) (Top) 2D slice through STA transverse view of T. spp. microtubule triplet, with insets showing position of pinhead (dashed green box) and A-C linker (dashed red box). (Bottom) Longitudinal 2D slice of STA centered on the pinhead (left) or A-C linker (right). (B) Transverse view of T. spp. microtubule triplet STA. Microtubule protofilament numbers are indicated, as are the pinhead and A-C linker (only the C-link is visible; the A-link lies on the edge of the volume and is thus less well resolved in this STA -for better view see STA centered on A-C linker in (D)). Prominent microtubule inner densities within the A-microtubule are highlighted (empty arrowhead next to A9, chevron next to A5), as are additional external densities at the A-B and B-C inner junctions (black arrowheads). Double arrowheads point to viewing direction in indicated panels. (C) Longitudinal view of T. spp. STA centered on the pinhead from the viewing point indicated in (B). The pinfeet (PinF1 and PinF2) and pinbody (PinB) are indicated, as are microtubule protofilaments A3 and A4. The average distance between pinfeet elements is 8.6 ± 0.4 nm and 7.9 ± 0.4 nm (N=3 each). Corresponding transverse views are shown below, illustrating the connection of PinF2 with protofilament A3. (D) Longitudinal view of T. spp. STA centered on the A-C linker from the viewing point indicated in (B). Microtubule protofilaments A8/9 and C9/C10 of two adjacent triplets are indicated, as are the connected A- and C-links. The average distance between A- and C-links is 8.4 ± 0.4 nm and 8.4 ± 0.3 nm (both N=6). Corresponding transverse views are shown below, chevrons point to connection.
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K-M. 5 days of MI-503 treatment impacts the expression of Pbk at protein level (K) and mRNA level (L), as well as ki67 mRNA levels (M) in human primary islets (donor number is four). qPCR data was from three independent experimets (n=3). **P = 0.0036 (L, 10 nM), **P = 0.0024 (L, 50 nM), **P = 0.0073 (L, 100 nM), *P = 0.0237 (M, 50 nM) (two-tailed unpaired student's t-test). ns, not statistically significant difference.
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(B) Western blot showing that K63-linked ubiquitination was required for RQC. Protein samples from UB-WT, UB-WT ltn1∆, ub-K63R, ub-K63R ltn1∆ cells expressing the GFP-R(CGN)12-HIS3 reporter were subjected to Western blot analysis using an anti-GFP antibody to detect the arrest products. Note the accumulation of RQC-specific CAT-tails in lane 2.
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(C) Related to (A). Calculation of minimum distances between pairs of FAS molecules as well as FAS molecules and their closest non-FAS neighbors in comparison to random distributions. Whereas FAS molecules are randomly distributed, their binders are not, confirming physical interactions. Supervised picking means that all single-particles were manually picked from the images. Randomized distance means that these manually picked particles were assigned random coordinates in each image (randomization of x,y coordinates considering image borders) and then their distance is calculated.
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C. Protrusion (Ps) and cell body (CB) fractions were isolated from cells treated with control-PMO or Rab13-PMO #2. The indicated RNAs were detected through nanoString analysis to calculate Ps/CB enrichment ratios (n=3; bars: mean ± s.e.m.). Note that only the distribution of Rab13 RNA is affected. **: p=0.01 by two-way ANOVA with Bonferroni's multiple comparisons test against the corresponding control. D. Levels of the indicated RNAs were determined using nanoString analysis from control- or Rab13 PMO #2-treated cells. (n=4; bars: mean ± s.e.m.). No significant differences were detected by two-way ANOVA against the corresponding controls.
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B-E Slice (B) and segmentations in three different orientations (C) of a large relaxed plate decorated with many nucleosomes on its right side. Slice (D) and segmentation (E) of a relaxed plate forming a tube decorated with nucleosomes. Data information: Scale bars: 50 nm insets, B-E)
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D. A schematic showing ponatinib inhibits multiple kinases involved in the SARS-CoV2-NTD-mediated cytokine signaling.
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E TET1 and TET2 RNAi in unmethylated BJAB cells. qRT-PCR for knockdown efficiency (top) and cell-surface staining for exon-5 containing CD45 isoforms (CD45RB, bottom) relative to control-transduced cells.
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F. MSD data for MB (endo) and cy3-pre-miR-181a-1 (exo) tracked particles were fitted with an anomalous diffusion model and α thus calculated (red). Total number of particles and axons analyzed: 67 particles, 20 axons (endo), 82 particles, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiment. Values are mean ± SEM. Abbreviations: MB, molecular beacon.
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(C) Proteasome inhibition rescues the mitochondrial import of mutant COA7 as well as the biogenesis and function of the respiratory chain.
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Wild type Balb/c mice were injected intra-peritoneally with solvent (daily) or 3 mg/kg chlorambucil (daily for 5 days) or 3.3 mg/kg cisplatin (daily for 3 days). Uptake of the apoptosis tracer 99mTc-Duramycin 2 h after intra-venous injection was quantified in selected organs using SPECT imaging in the indicated organs. Representative maximum intensity partial projections showing tracer distribution are shown.
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(D) Localization of VgpA (upper panel) or in VgpAL10A (lower panel) in HeLa cells after transfection. EBP2 was detected with anti-EBP2 antibody (red), VgpA and VgpAL10A were detected with anti-VgpA sera (green) and nuclei were stained with DAPI (blue). White arrows indicate co-localization of EBP2 and VgpA (upper panel). VgpAL10A localizes
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(B) An aliquot of fractions VV and SV was treated with proteinase K either in the presence or absence of Triton X-100 as indicated. Because of the presence of protease inhibitors in the vesicle isolation procedure, an intermediate-sized API degradation product results from proteinase K treatment. The V fraction represents one-tenth of the load of the Optiprep gradient. Proteins were detected by Western blotting. The positions of prAPI and mALP are indicated.
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C Confocal images showing the basal region of the organ of Corti cultures treated with medium alone, 10 µM KU55933, 10 µM CDDP or 10 µM CDDP in combination with 10 µM KU55933 for 5 days and immunolabeled for Myosin 7A (red). Scale bar=24 µm.D Histograms representing the numbers of surviving IHCs (red bars) and OHCs (blue bars) for all treatment conditions after 5 days (n=5 cochleae per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (*P=0.03, **P =0.004, ***P ≤ 0.0008, CDDPvs. control, or CDDPvs. CDDP+KU55933).
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HEK293 cells: (B) Representative immunoblots (of three independent experiments) with antibodies against HA, the 3G10 epitope (for the HS stub) and V5.
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E LC3 and WIPI2 were co-stained and imaged using an LSM700 confocal microscope. Arrowheads point at co-localized LC3 and WIPI2 puncta. Scale bar: 5 µm
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(B) Graphical representation of the SPOP concentration of each oligomeric species within a 27 µM SPOP28-359 sample. The cartoons' sizes are scaled based upon the fraction present within the total sample.
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B Spaghetti plot showing fold change radiance over time, individual xenografted mammary glands (light grey), averaged control samples from individual xenografts (dark grey), average values of control samples from all xenografts (black, red dots), n=12-224 xenografted glands per time point.
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(F) Induction of autophagy by the TBD. HeLa cells stably expressing GFP-LC3 were transfected with pcDNA3.1 or constructs encoding the indicated BCN1 variants. After 24 h, Flag‐tagged proteins were detected by immunoblotting and the frequency of cells with >5 GFP-LC3+cytosolic puncta (GFP-LC3VAC cells) was assessed (mean values±s.d., n=3; *P0.01, **P0.001 versus pcDNA3.1‐transfected cells).
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(K) Proteome analysis confirms sphinganine-induced disruption of (epi) genomic integrity (n = 3).
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WT, Abro1−/− and Nlrp3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. Flow cytometric analysis of peritoneal cell exudates (B). n = 6 for PBS groups and n = 12 for MSU groups.
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Kaplan-Meier curve of 3.5% DSS-treated Ang-deficient mice (Ang-/-, n = 14) or littermate controls (WT, n = 13).
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Mapped views showing the RNA-seq and ChIP-seq results of four male genes in DNA replication and DNA repair pathways.
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(e) Mitochondrial DNA content was assessed by real-time PCR. Results are shown from six-independent experiments.
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A. Representative spinning disk confocal live-cell imaging time series images of U2OS PA-GFP-α-tubulin cells conditionally co-expressing KIF4A shRNA and RNAi-resistant mCherry-Kif4A variants induced using doxycycline. Chromosomes were stained using SiR-DNA. White arrowheads highlight poleward flux of the photoactivated regions. Scale bar, 10 µm. Time, min:sec.
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B) Significance values from transcription factor pathway-focused GSEA of GTx-024-treated tumor-free versus tumor-free control transcriptomes. *FDR< 1e-5 was determined for the CTNNB1 gene set, and was set to 1e-5 for the plot.
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Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer. (B) Vacuole morphology was assessed as in Fig. 3C. Scale bar: 5 µm. (C) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments.
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Fold increase in Oct4-GFP-positive cell colonies in the above cells. Data are shown as mean + s.d. (n=5); ** indicates P-value < 0.01; *** indicates P-value < 0.001.
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D. Boxplots of EBs diameter at day 10 of EB formation under M15, CM, and M15 supplemented with Bmp4 conditions. Each boxplot was plotted for more than 10 EBs. EBs cultured under CM or Bmp4 condition were compared to EBs of the same cell line cultured under M15 condition. * P < 0.05, ** P < 0.01.
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M, N GlcNAc recognition of FBXO2 required for GAS xenophagy. Non-transfected HeLa WT cells and HeLa FBXO2-KO cells transfected GFP, GFP-FBXO2, GFP-FBXO2 YW/AA were infected with GAS WT for 4 h, fixed, and immunostained for ubiquitin. Percentages of cells with LC3 (M) or ubiquitin (N) -positive GAS were quantified.
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I: Quantification of immunoblots revealed moderate and substantial PIKfyve loss in Type-1 and Type-2 CJD, respectively (unpaired t-test). **: p<0.01; ***: p<0.001. Error bars represent s.e.m.
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(H) Synergy score for combinations of serial dilutions of PI3Kβi (AZD8186) plus EGFRi (gefitinib) tested on six PTEN-null and five PTEN-WT TNBC cell lines for 6 days in three independent experiments. The score was obtained analysing the viability data through the software Chalice Analyser. Mean of the synergy scores ± SD. Statistical significance of Mann Whitney two-tailed test *P=0.0303.
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(B) Ndc80-9D is defective for MTOC sorting. The density map of MTOCs along the spindle axis in Zp3-Cre Ndc80f/f oocytes expressing Ndc80-WT, Ndc80-9D or Ndc80-9A. The color represents the percentage of MTOC volume coded from dark (0%) to white (50% or more of the total volume).
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(I) Ingenuity Pathway Analysis (IPA) of transcriptome in gas-1(fc21) vs. wt (top panel) and gas-1(fc21); cest-2.2 O/E vs. gas-1(fc21) (bottom panel) with their predicted consequences on lipid metabolism. In red/green: results from the transcriptome. In blue/orange: IPA-generated predicted outcome of this gene dysregulation. Shades of red and green are proportional to log2(fold change), while shades of blue and orange are proportional to the statistical strength of the prediction (z-score), as calculated by IPA.
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Assessment of transcription levels measured by EU labeling assay upon treatment with DHS on hCMs
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(F) Representative immunofluorescence images of mitotic neuroblasts from squashed brains showing distinct chromosome content. Spc105 was used as reference for KTs. Chromosome content is shown for each neuroblast.
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Representative membrane of a mitochondrial protein synthesis experiment. The results were normalized to the gel loading
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B. HEK293T cells were transfected with indicated constructs and immunoprecipitation (IP) was performed using GFP specific antibodies and probed for HA-AGO2 by western blotting (WB) using HA antibodies. Endogenous RanGAP and Ubc9 were probed with specific antibodies.
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Upon being complexed with sgRNA, SaCas9 confers stable binding to the DNA target when 6 PAM-proximal matches exist. It then triggers sequential DNA unwinding from the PAM-proximal region to the PAM-distal end and samples adjacent to the DNA for guide RNA complementarity. More than 18 bp RNA-DNA matches close to the PAM allow complete R-loop formation and endonucleolytic cleavage of both DNA strands. Afterward, SaCas9 remains bound to the PAM while autonomously releasing the PAM-distal DNA. The interplay between SaCas9 and the DNA is mediated by the post- and pre-PAM interactions, as indicated by the blue and black triangles, respectively.
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C Word cloud representing the Gene Ontology Biological Process terms enriched (Benjamini-Hochberg corrected p-value <0.05) in the lists of genes up-regulated (UP) or down-regulated (DOWN) upon lnc-SMaRT knockdown. The size of the words correlates with the Benjamini-Hochberg corrected p-value.
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Analysis of viability of SNU410 pancreatic cancer cells assessed 168 h after transfection with independent non-targeting siRNA (three different duplexes used, siCTRL#1, #2 or #3) or targeting VPS4A (duplexes #2, #4 or #5), VPS4B (duplexes #1 or #2) or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of 3 independent experiments ±SEM. All values were normalized, cell viability of averaged (avrg) siCTRL#1-, #2- and #3-transfected cells was set as 100%. Two-tailed unpaired t-test; ****p<0.0001.
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F) CCK8 assay of SNU719 and SNU-4th cells to determine cell proliferation under the indicated culture conditions (10% FBS or 0.1% FBS).
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ATPase stimulation for GFP-tagged Rad54, Rdh54NRad54, Rdh54 or Rad54NRdh54 in the presence of Rad51-ssDNA. Error bars represent s.d. of three independent experiments.
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 C mRNA expression in female Lin-Sca1+ cells, differentiated in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT-PCR (n=3) t-test Cited4-/- vs. Cited4+/+ (Rosi) *P=0.013 (Ucp1), **P=0.004 (Cpt1b), *P=0.026 (Dio2)
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A) Immunoprecipitations and Western blots were performed on MEFs stimulated or not with IFNγ (100 U/ml) and infected with an RH strain expressing type II GRA15-HA and as a control RH expressing GRA45-HA. The blots using antibodies against TRAF6 and TRAF2 were made after stripping the first blot. Left panel and right panel were run on a single gel; vertical white lines indicate excision of irrelevant lanes.
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B. U2OS cells treated with HU in the presence or absence of ATR inhibitor (ATRi) were fractionated and immunoblotted with indicated antibodies. Relative RADX levels on chromatin were quantified and normalized to histone H3. See also Fig EV2A.
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Experimental design for TSLP neutralization in vivo. TSLP neutralization was performed by triple intra-dermal injections of 20 μg of anti-TSLP or IgG to the right ears of DKO* and DKO*15 mice five days after of psoriasis-like induction. EdU was added into mice by IP injection 2 hour before euthanasia.
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(C) Left panel, western blotting with HeLa and TRIM16KO cell lysates probed with indicated antibodies. Right panel, densitometric analysis (mean ±SD) of protein band intensity relative to actin, n=3, ***p < 0.0005 (Student's unpaired t-test).
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D. TBK1 is required for EGF-stimulated mTORC1 and mTORC2 signaling: TBK1+/+ and TBK1-/- MEFs were stimulated with EGF as in A.
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(c) STM of 30-d-old (30 d) Spd1mM+ and Spd5mM+ and Spd− flies (n = 8-9 independent experiments, F = 14.48, one-way ANOVA with Bonferroni correction).
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BNIP3 levels in primary melanoma and metastases within the SKCM TCGA cohort, using Mann-Whitney's non-parametric test. The box represents the median and the 25th-75th percentile with the whiskers representing the minimum and maximum values.
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C. The results of the luciferase reporting system showed that LncBMP4 could rescue the gga-mir-12211 inhibitory effects against BMP4-3'UTR (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, **** p < 0.0001, one-way ANOVA).
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A. Unspliced RPL28, an NMD substrate, was enriched in Nmd4-TAP purification in comparison with a control untagged strain, as measured by reverse transcription followed by quantitative PCR. Bars correspond to the mean of pre-RPL28 enrichment for 3 independent experiments, as compared with RIM1, a non-NMD mRNA. Error bars correspond to SD. The indicated p-value was computed using the Welch t-test, single-sided comparison
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(C) Prediction of population-wide, between-line, wing size variations from the mediator-linked genotypes. Mediator-linked eQTLs relevant for each condition (size traits, sexes) were used to predict wing size traits (absCS/relCS) of 143 DGRP lines for each sex. Spearman correlation coefficient and the P-value, and R2 from linear fitting are shown for each case (size traits, sexes). The dark and light grey zones indicate 50% and 95% prediction ranges.
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Overexpression of Inh1 decreases ∆ψm in ρ0 cells. Cells were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid-log phase. Cells were then stained with 125 nM TMRM for FACS analysis.
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Indirect calorimetric measurement (n=10/group) of (H) heat productio at P77 Bar graphs represent mean±SD The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs
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D Correlation between gene expression and basal cell adhesion to VN and reduction in cell adhesion upon Plg treatment. Adhesion 4 h after seeding the cells on VN (expressed as cell index) was measured in the experiment described in panel C. The extent of cell adhesion reduction was calculated at the indicated time point in panel C as percentage of the cell index value measured in control wells treated with vehicle. Both parameters were correlated with the expression levels of the indicated genes. Gene expression data were downloaded from the CellMiner web tool. The table shows gene name, Pearson correlation coefficient (r) and p-value.
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(D) Ubiquitination of AGIA-PLZF in AGIA-PLZF and FLAG-CRBN expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) in the presence of MG132 for 10 h. AGIA-PLZF was immunoprecipitated using anti-AGIA antibody and the polyubiquitin chain on AGIA-PLZF was analysed by immunoblot.
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C Comparison of the cryo-EM structure of H2A.B-NCP with the structure of canonical nucleosome in gyre view. Arrows indicate the direction of H2A.B-NCP DNA movement during nucleosome gaping transition. The dashed lines indicate the ~10 degrees rotation of H2A.B DNA at SHL 5. H2B N-tail refers to H2B residues 26-32 which are exclusively observed in the canonical nucleosome structure. Histones H2B in H2A.B-NCP are colored in warmpink, Their counterparts in canonical nucleosome are colored in red. H2A.B-NCP DNA in green is shown in surface mode for clear comparison.
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(A) Schematic illustrates the size and location of the retinal explant (dashed line).
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Representative immunofluorescence of primary cilium in WT or Mecp2 null MEF cells. Cells were starved for 48 h before staining with anti-γ-tubulin (green) and anti-acetylated α-tubulin (red) antibodies. Merge of all channels and DAPI staining (blue) is depicted for both genotypes. Scale bar 20 µm, and 10 µm in the enlarged image. Arrows indicate not ciliated cells.
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A. Total protein copy number of the oxidative phosphorylation complex I-V in bulk liver tissue (n=6 independent biological replicates) and primary cell types (isolated primary cells from n=3 individuals for each cell type). Values are presented as mean ± s.d. B. Protein copy number for members of the oxidative phosphorylation complex I-V in hepatocytes and Kupffer cells with Pearson correlation coefficient indicated.
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G RBM47+/+ and RBM47+/- mice were infected with HSV-1 (1× 107 pfu) via caudal vein injection. Viral genome DNA and progeny titers were tested using blood samples from infected mice. Data are represented as the means ± SD of n = 6 biological replicates. H qRT-PCR analysis of IFNAR1 and Cig5 mRNA in blood samples from HSV-1-infected mice. The PCR results are represented as the means ± SD of n = 6 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).
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D. NH1 cells containing the integrated HIV-1 LTR-luciferase reporter and expressing Tat were transfected with siDKC1 or siNT. ChIP-qPCR analysis was performed to determine the levels of the indicated factors bound to the HIV-1 promoter. The signals were normalized to those of input and plotted.
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Schematic model of the differences between a cell expressing one or two actins to perform two cellular functions. (Top) A model of the molecular mechanisms by which two actin isoforms may segregate to different actin networks. On the left, a system carrying wild-type actin is able to generate both the branched- and linear-networks. On the two central panels, defective interactions of an actin isoform with one or several ABPs, affect branched- or linear-network assembly. On the right, combining these two actin variants in one cell should trigger a natural segregation of actins and rescues the wild type actin organization. (Bottom) Effect of perturbing an actin assembly pathway for cells using one or two actin variants. On the left, when one actin is shared for two actin functions, the inhibition of one actin assembly pathway (for example branched-networks with CK-666) leads to a reinforcement of the other actin assembly pathway. On the right, when two actin variants are used for two different actin functions, this effect is limited as both actin networks assemble more independently. In other words, having a system with two actin variants can buffer against the addition of the drug.
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B The Na+ and K+ contents and Na+/K+ ratio in roots and shoots of three groups of 369 accessions after treatment with 150 mM NaCl for 1 d.
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c, IpaJ or its catalytic mutants were expressed from a galactose-inducible promoter (pGal413 vector) and assayed for a growth arrest phenotype on galactose or glucose (control) carbon source.
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(E) Analysis of differential glycosites identified with the TMT labeling approach using the Qluqore Omics Explorer software. The heatmap represents O-glycosites most significantly contributing to sample variation, using ANOVA with a q value cutoff of 0.1, where each compared sample consists of three individual KO clones. Each horizontal line depicts the median quantification of glycopeptides contributing to the same glycosite. Alternative differentially glycosylated site positions are separated by semicolons on sites derived from glycopeptides with multiple HexNAc/HexHexNAc modifications.
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I Immunoblotting using an anti-P-Akt, anti-P-S6 or anti-β-actin (loading control) antibody of protein extracts from SOM230-treated or SOM230-untreated CAF incubated or not with IL-6 neutralizing antibody (representative of n = 3).
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(G) SHP2 shRNAs #1, #2, #3 reduce BMEC, not B16F10 cell proliferation; representative of 3 experiments; error bars: ± S.D.; 5 replicate cultures/experiment.
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quantification of hypocotyl lengths (G) of 4-day-old seedlings grown with or without supplemental UV-B. The scale bar for all lines represent 5 mm except for cop1-5 where the scale bars represent 1 mm. Violin and box plots are shown for n > 60 seedlings; upper and lower hinges correspond to the first and third quartiles, the horizontal line in the interior of the box indicates the median. Data Information: lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.
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(E) (I) Percentage of neutrophils with LC3B punctuated structures. E. coli (bar 1: 84±2.2%) compared with E. coli+3‐MA (bar 2: 4±1.95%). Data are representative of six independent experiments and presented as mean±SD. Wilcoxon matched‐pairs test, *p<0.05. (II) Formation of LC3B puncta in phagocytosing neutrophils and (III) in cells treated with 3‐MA before incubation with opsonized E. coli, as observed by immunofluorescence confocal microscopy (1. LC3B: red; 2. FITC conjugated E. coli: green; 3. DAPI: blue; and 4. merge). Solid line arrows point the colocalization of LC3B puncta with E. coli bacteria. Dashed line arrows point LC3B aggregates that are not colocalized with E. coli.
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B H3K9me3 immunostaining in the indicated HepG2 cell lines with nuclear counterstaining in DAPI. The indicated areas are zoomed-in for a better observation of the H3K9me3 signal profile. All images are maximum intensity Z-projections of confocal stacks.
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Representative immunostaining of Arl13b (red) and γ-tubulin (green) in the P14 mouse cortical layers. Nuclei were stained with DAPI (blue). Scale bar 10 µm.
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(c) CD39, Atg5 and as a control β-actin protein expression in isolated Atg5fl/+, Atg5fl/fl, KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes. Protein lysated were analysed at 4 days after AdCre infections by western blot. Quantifications of the CD39/β-actin ratios are shown for each lane, normalized to the Atg5fl/+ samples. Two independent blots are quantified.
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D: Top: Basal area (black line) and total force F (blue line) as a function of time, for the same cell as in A. Inset: Temporal cross-correlation function (CF) of the area and F (blue) and of the area change rate and F (magenta). Bottom: Kymograph of T along the cell outline.
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Immunofluorescence stainings of PNRC1 (red) and Ki-67 (green) performed on a malignant lymphoma (D). DAPI was used to stain cell nuclei. Scale bars:50µm.
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LN229 were transfected with siNT, siNoxa 1, siNoxa 2 or BAK-siRNA for 72h. Thereafter, cells were treated with the combination treatment of 1 µM ABT263 and 20 µM LXR623 for another 24h. After conclusion of the treatment, cells were harvested, fixed, stained with propidium iodide and analyzed by flow cytometry for DNA - fragmentation.
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B Plasma total cholesterol levels in mice that received adenovirus GFP (control) or PLTP. Blood and tissues were harvested seven days after adenovirus infection. n=8 in mice on RD and n=7 in mice on HFD for 14 weeks. ***P<0.001 relative to GFP by Student's t-test.
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