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Genome-browser view of the Gata6 locus showing accessibility and chromatin mark changes in indicated mutants, and of ChIPseq signals of indicated TFs and H3K27ac in E6.5 VE and EPI. The grey area indicates enh-45.
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(B) PI4KB and c10orf76 form a stable complex. The complex of c10orf76-PI4KB eluted from a S200 superdex 10/300 GL increase gel filtration column (GE) at a volume consistent with a heterodimer (169 kDa), while c10orf76 alone eluted at a volume consistent with a monomer (79 kDa). Lines with MW values indicate elution of MW standards (158 kDa aldolase, 75 kDa conalbumin).
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H Plasma insulin level (µU/mL) of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls at 180 min after IPGTT (n = 4 each). Mean ± SD.
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A. Quantitative RT-PCR detection of CAV1, CAV2 and CAVIN1 in primary human umbilical venous endothelial cells (HUVECs) upon Notch blockade (dnMAML) and induction (NICD). n=3, data represent mean ± SEM, unpaired t-test.
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A, B The resident/intruder experiment showed that the attack frequency (A) and duration (B) of the miR-200b/a+REST DKD mice were dramatically improved compared with those of the miR-200b/a KD mice [200 KD: miR-200b/a KD; (200+R) DKD: (miR-200b/a+REST) DKD; NC: n=10 mice, miR-200b/a KD: n=9 mice, miR-200b/a+REST DKD: n=13 mice; data represent the mean±SEM; A: F=10.62, B: F=10.54, ***p<0.001; one-way ANOVA and Bonferroni pairwise comparisons].
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Whole-plant recordings of changes in CO2 assimilation (A) from self-grafted donor lines (WT/WT, stp1stp4/stp1stp4) and reciprocal grafting of shoot/root (WT/stp1stp4, stp1stp4/WT) plants. Data shown are means ± SEM; n = 3 per genotype.
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Na+/K+ ratio contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4).
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(A-E) Cbl expression in germaria. (A) Immunostaining of wild-type germaria with anti-Cbl 8C4.
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F, IF analysis of A549 cells transfected with a Myc-tagged wild-type USP7 (Myc-USP7-WT) or three Myc-tagged truncated mutants. Representative images are shown (magnification, ×40).
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(C) Cells were transfected with siRNAs as indicated, followed by incubation in serum-starved media for 24 h, and immunostained for ARL13B (red). Scale bar, 10 μm. The bar graph represents the quantification of the percentage of ciliated cells. Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.
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D. Detection of ubiquitylated Sufuco-immunoprecipitated from HEK293T cells co-transfected with HA-tagged Sufu, Myc-ubiquitin (Ub) along with either Flag-tagged Fbxl17 wild type (WT) or a mutant lacking the F-box domain (Fbxl17ΔF). MG132 (10µM) was added in all samples.
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(B) HeLa cells were starved or not starved for 2 h and immunostained for SNX18 and GM130. Panels to the right show enlarged views of the boxed regions.
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(C) UV sensitivity assayed by 10-fold serial dilutions of different mutant combinations with rad3-102 allele on YPAD plates after exposure to the indicated UV-C doses. Four biological replicates have been performed.
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(b) YAP/TAZ deficiency induced a reduction of ATF4 protein level and nuclear localization. HLE cells were transfected with siCtrl or siY/T, and ATF4 and YAP/TAZ were visualized by immunofluorescence microscopy. DAPI visualized nuclei. Scale bars, 50μm. Results represent three independent experiments.
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D. Histograms reporting the percentage of ShtxB or ChTxB positive cells in each condition as in Fig. 1F (data are means ± SD of at least 3 independent experiments; *p <0.05, ***p <0.001 [Student's t-test]).
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(E) Pie chart showing the outcomes of CAR T cell-tumor contacts. Contacts were classified into 3 categories: no calcium elevation in the target (no hit), transient calcium elevation (sublethal hit), prolonged calcium response in the target (putative lethal hit). Data were pooled from three independent experiments.
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(d) Proteinase K protection assays performed in the presence or absence of Triton X-100 to disrupt peroxisomes on equal masses of peroxisome fractions (HEK293 cells) collected at the indicated time points. The lysates were immunoblotted for TSC2, TSC1, Rheb, catalase and PMP70. Uncropped images of western blots are shown in Supplementary Fig. S6.
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HEK293T cells stably overexpressing wild type ATF6α or a D564G mutant were treated with 10 mM DTT as indicated to induce ER stress in the presence of the S1P inhibitor. Cell lysates were prepared, separated by SDS-PAGE and then probed with anti-ATF6α. Blot reveals the presence of O-linked glycan modified Golgi-translocated ATF6 (designated G), which is absent for the D564G mutant, confirming a lack of ER to Golgi trafficking.
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(E) Macrophages were treated for 24 h with CL097, ssRNA40, and rapamycin in the presence of 45 nmol/L or 100 nmol/L 25D3 and lysed. Immunoblots of LC3B isoforms and SQSTM1 using antibody to LC3B, SQSTM1 or β-actin. Bar charts are reported as mean ± s.e.m., n = 4. * p<0.05.
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(B) Cross-presentation experiments to exclude release of NeoR and re‐uptake as exogenous protein. EBV‐B1.25 cells transfected with the neoR expression plasmid pINCO‐NeoR, were co‐cultured for 24 h with the same number of untransfected EBV‐B1.11 cells, or vice versa. Then, T cells were added and co‐cultured with the cells for additional 24 h. To test for NeoR release by neoR‐transfected EBV‐B1.11 cells, total supernatant (200 μl) of 1×105 EBV‐B1.11 cells was harvested 48 h post transfection by centrifugation, and was used as culture media for 1×105 untransfected EBV‐B1.11 cells. After 48 h, 1×105 20-4/A4 T cells were added and GM‐CSF concentration determined after additional 24 h.
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(C) Immunofluorescent staining of cleaved APPC586 (red) and Aβ (green) in cerebral cortex of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. (D) Quantitative analysis of cleaved APP C586 positive cells and Aβ positive cells, respectively. The density of both cleaved APP C586 positive cells and Aβ positive cells were significantly increased by colonic-injection of AD brain extracts and decreased through vagotomy before colonic-injection. Representative data of three samples, data are shown as mean ± SEM. ****P<0.0001, one-way ANOVA.
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(D) Volcano plot of differentially phosphorylated proteins in gas-1(fc21) mutants compared to wt. Cutoffs: 20% FDR and |log2(foldchange)| >0.345 (in red).
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C. Morphological observation of the number of embryoid bodies in the PGC in vitro induction model after the overexpression and interference of gga-mir-12211. BMP4 induction was regarded as control. Scale bar: 60 µm (n = 3 independent experiments).
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(C, D) Parental (WT) RPE1-hTERT p53-/- Cas9, RNF8-/-, and RNF168-/- cells were treated with either a non-targeting siRNA pool (CTRL) or a pool targeting RAP80. 48 h post-transfection cells were irradiated (2 Gy) and processed for immunofluorescence 1 h post-IR treatment using antibodies against BRCA1 and γH2AX. Quantitation of the percentage of cells with > 10 BRCA1 foci that colocalize with γH2AX is shown in (C). A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates). Representative micrographs are shown in (D). All scale bars are 5 μm.
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A Schematic setup of the screen for PD-L1 regulators in the presence and absence of IFN-γ. RKO reporter cells expressing PD-L1-eGFP fusion protein (blue) were mutagenized with an sgRNA/Cas9 library. PD-L1low cells were enriched by FACS and sgRNA target genes were identified by deep sequencing. An enrichment analysis determined frequencies of different sgRNAs and their target genes.
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(I, J and K) Polyphenols inhibits the virulence levels and in planta growth of Psph in bean plants. Bacterial virulence levels were estimated by inoculating Psph strains with or without polyphenols onto host bean plants. Disease symptoms were recorded 5 days after inoculation. Leaf disks (1 cm2) were removed 5 days after inoculation and diluted in sterile water to count bacterial CFUs. The experiment was repeated three times. Data information: , * indicate significant differences between mock group and strains under the different test conditions as indicated. (Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001). Error bars represent standard deviations (n=3 biological replicates).
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Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) +/- FGF7 (10 ng/ml), added at the indicated time points 12 h after infection, , DNA samples were analyzed by qPCR for Glyc-B DNA relative to ACTB (C),
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(D) Cells were treated as described in (A) and cell extracts were applied to size exclusion chromatography and the ferritin content, in selected fractions, was determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.
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A) Mfn2protein expression was measured in gastrocnemius muscle from young (6-month-old) and old (22-month-old) mice (n= 11-13 mice per group, *p<0.05) Data were normalized by tubulin and expressed as relative values of young mice.
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Genes down-regulated in nuclear RNA-seq after CDK12 inhibition have higher relative occupancy of P-Ser2 in their gene bodies. Metagene analyses of P-Ser2 ChIP-seq data Each transcript was divided into two parts with fixed length (transcription start site (TSS) -3kb to +1.5kb and transcription termination site (TTS) -1.5kb to +3kb) and a central part with variable length corresponding to the rest of gene body (shown in %). Each part was binned into a fixed number of bins (90/180/90) and average coverage for each bin was calculated for each transcript in each sample. The curve for each transcript was normalized to a sum of one and then averaged first across genes and second across samples. Dotted lines indicate: TSS, 1500 nucleotides downstream of TSS, 1500 nucleotides upstream of TTS and TTS. The color track at the bottom of each subfigure indicate the significance of paired Wilcoxon tests comparing the normalized transcript coverages for each bin between untreated (CTRL) cells and cells treated with 3-MB-PP1. P-values are adjusted for multiple testing with the Bonferroni method within each subfigure; color code: red=adjusted p-value≤10-15, orange=adjusted p-value≤10-10, yellow=adjusted p-value≤10-3.
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C. The L. monocytogenes EGD-e wild-type strain (WT), the ΔinlK mutant (ΔinlK) and the complemented strain (ΔinlK+pPL2 inlK) 104 CFU were inoculated i.v into BALB/c mice. Animals were euthanized 24 h, 48 h, 72 h or 96 h after infection and organs were recovered, homogenized, and homogenates serially plated on BHI. The number of bacteria able to colonize liver (left panel) and spleen (right panel) is expressed as log10 CFU. Four animals per bacterial strain, per time points and per experiment were used. Statistical analyses were performed on the results of 3 independent experiments using the Student t test. P values of <0.05 were considered statistically different and are labeled here as *.
|
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J. Representative axons. MB puncta are indicated (white arrows). Dashed white lines delineate axons. Abbreviations: MB, molecular beacon. Scale bars: 5µm.
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IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells transfected or not with GFP-Polη. >180 (track length) and >400 (origin frequency) DNA fibers obtained from 2 independent experiments were measured for each condition.
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Results of the ELISAs used to determine IL6 (B) secretion in PMA-differentiated THP-1 cells transfected with siCtrl, siRNF34-1, siRNF34-2 or siRNF34-3 oligos and infected with VSV for the indicated times. ELISA data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
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A Cartoon of constructs containing ChHFR1 or AtHFR1 under the HFR1 promoter of A. thaliana (pAtHFR1) used to complement hfr1-5 mutant of A. thaliana (At hfr1-5).
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Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Representative multicolour flow cytometry analysis of 5 independent experiments showing CD11b and CD45 positive populations in single viable cells in saline or LPS-injected mouse brains.
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Western-blot analysis of p-AKT, p-S6, and p-p70S6K in total protein lysates from GSC#9 treated for 1 hour with vehicle (DMSO) or 20 μM of phenothiazine compounds (MPZ, FLU, CHLO, and CYA). Total AKT, total S6 and total p70S6K served as loading controls.
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N. JunD overexpression in PIME cells suppressed Pbk protein levels (M, Lane 2 vs Lane 1), but did not affect Pbk expression in menin-null PIME cells (M, Lane 4 vs Lane 3).
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HeLa cells were transfected and treated The immunofluorescence intensity ratios of centromeric EGFP-TOP2A/ACA (D), centromeric EGFP-TOP2A/arm EGFP-TOP2A (E), and arm EGFP-TOP2A/DNA (F), were determined on around 160 chromosomes in 8 cells. Data information: Means and error bars representing S.D. are shown unpaired t-test).
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(D) CCM1 incompatibility is symmetric between S. cerevisiae and S. bayanus. The CCM1 ORF in S. bayanus was replaced by either Sc-CCM1 (Sb+Sc-CCM1) or Sb-CCM1 (Sb+Sb-CCM1, as a control) coding regions. Only the Sb+Sc-CCM1 strain exhibited respiration defects and the defect could not be rescued even when a whole set of S. cerevisiae chromosomes were provided (Sb+Sc-CCM1 x Sc-ρ⁰).
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(A) Quantitative RT-PCR of Chrne, Musk, Lrp4, and Ctgf in C2C12 myotubes treated with 10 ng/ml BSA or recombinant human CTGF.
|
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, MARCKS (C) expressions were quantified in cells stably expressing dCas9/KRAB/BFP and the NC or sgRNAs.
|
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A Regulation of svh-2 expression in response to axon injury. Axon injury initiates cAMP signaling and the Ca2+-p38 MAPK pathway, which together function to induce the formation of an ETS-4-CEBP-1 transcription factor complex. This complex binds to the svh-2 promoter to induce svh-2 expression. MXL-1 forms a complex with MDL-1.
|
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AAV-eGFP (eGFP) or AAV-hTau-eGFP (hTau) (1.13×1013 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) plus hTau was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, learning and memory were detected by MWM test. (C-E) Overexpression of Y701F-STAT1 mitigated hTau-induced spatial memory impairment shown by the decreased latency to reach the platform (C), the increased crossing time in the platform site (D) and time spent in the target quadrant (E) measured at day 6 by removed the platform (n=7-10 each group). Data information: Data were presented as mean ± s.e.m , two-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.
|
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] |
(H, I) Mtb growth rates of C3H BMDMs transfected with miR-342-3p mimic (H), or B6 BMDMs transfected with miR-342-3p inhibitor (I) after Mtb infection. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.
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(A) tRNA level measured by RT-qPCR in asynchronous (async.) culture and after 90 min on HU in the indicated strains. Expression is normalized to ACT1. The repression is expressed as a ratio of HU treated/asynchronous cells in percent.
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Immunohistochemistry images and quantifications of Ki67 in OVCAR4 xenografts in mock (N = 4), carboplatin (N = 4) and carboplatin+BI-D1870 (N = 5) treated mice. Scale bars: 100 μm.
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B. DNI of ΔPrP with a deletion in the C-terminal part of H1∼H2. Δ171-175 (see Fig. 7A) was transiently expressed in N2a cells (left panel) and DNI efficiency evaluated in 22L-ScN2a cells (right panel) (mAb 3F4). Despite the much shorter deletion size, Δ171-175 exerted only inefficient DNI, similar to Δ159-175 (right panel).
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A: PAI-1 immunohistochemistry (upper panels) and quantification of PAI-1-positive glomeruli (lower panel) in kidneys of PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 4 for PAI-1flox and PAI-1∆endo mice, respectively. B: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 12 for PAI-1flox and PAI-1∆endo mice, respectively. C: TUNEL assay (upper panels) and quantification of TUNEL-positive glomeruli (lower panel) in kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 6 for PAI-1flox and PAI-1∆endo mice, respectively. D: WT1 immunostaining (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 4 and n = 6 for PAI-1flox and PAI-1∆endo mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: PAI-1∆endo versus PAI-1flox mice.
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(C) Cellular thermal shift assay (CETSA) of different targets of the 144DG11hetero-assembly (B). Only LAMP1 was significantly protected by 144DG11 from heat-mediated denaturation (see right shift in its Tm in the lower panel) suggesting its specific interaction with 144DG11. n=3 biological replicates. Error bars represent S.D.
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(B) Two representative biological functions of ZBTB7B: 1) changing target gene expression; 2) recruiting target proteins for ubiquitination. CUL3: Cullin3, E2: ubiquitin-conjugating enzyme, SUB: substrate.
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Gene expression arrays were performed in 6 week old Foxd1Cre::Pdgfrb+/J mice compared to wt mice. Listed are genes with log2 fold changes higher or lower than 0.6. Out of these 46 genes, 17 are IFN-regulated genes, 5 are involved in autophagy and 5 in matrix remodeling and fibrosis. Same genes were analysed in microdissected glomeruli (G) and tubulointerstitium (T) of patients with diabetic (DN, G: n=14, T: n=18), hypertensive (HT, G: n=15, T: n=21) nephropathy and living donors (LD, G: n=42, T: n=42) as controls, revealing a similar expression profile.
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(b) Left: electron microscopy of starved C38 and IB3-1 cells. The right-hand images are higher magnifications showing areas enriched in autophagosomes in C38 cells (arrows) and perinuclear mitochondria in IB3-1 cells. N, nucleus. Scale bar, 250 nm. Right: numbers of autophagosomes per cell counted in 20 cells per experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus C38 cells; ANOVA.
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A DNA accessibility (ATAC-sequencing) and chromatin marks (ChIP-seq) signals in the Celf2 region (+/- 50kb) using the UCSC genome browser. ATAC-seq peaks called in the two conditions are reported below each track. The differential peaks specific for one of the two conditions are reported in the tracks ATACseq_specific peaks_WT and ATACseq_specific peaks_GS∆44. ChIP-seq signals for H3K4me3, H3K27ac and H3K27me3 in WT and GSΔ44 myoblasts (WT myoblasts in light orange; GSΔ44 myoblasts in light blue). ChIP-seq for MyoD in control cells was obtained from (MacQuarrie et al, 2013) (GSM1218849). Orange boxes highlight the regions containing the first exon of each isoform (a, b and c).
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HepG2 cells were transfected with lentivirus vectors expressing shRNA targeting KLF10 or negative control (Ctrl). n=3. Results represent three independent experiments. (C) CD36 protein expression the plasma membrane fractions, and its normalization to ATP1a1. Data information: ** P < 0.01. Results are shown as mean ± SD. Student's t test C).
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A. Scheme representing the strategy used to identify protein interacting with BAZ2A on chromatin (Chromatin-IP).
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(a) Genomic deletion of Atg7 in H-2Db-gp33+ T cells and in total activated (CD44hiCD62Llo) CD8+ T cells from Atg7fl/fl mice (Cre −) and Atg7fl/flGzmb-Cremice (Cre +) at day 8 after infection with LCMV Armstrong strain; results are presented relative to those of Atg7fl/fl mice, set as 100%. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.).
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(N) Sum of TLC-separated FFA of 5-day old animals (n=5 biological replicates). (O) Quantification of individual FFA detected after TLC separation in 5-day-old gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes (n=5 biological replicates, n.d.= not detected). Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.
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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 Representative heatmaps are shown in (G).
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(F) Increased bacterial growth of Pst DC3000 hrcC in ddl-6. Leaves of four-week-old plants were inoculated with Pst DC3000 hrcC at OD600=5x10-4.
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(b) Overlay of 2D-[15N, 1H]-TROSY spectra of BakΔTM with (red) and without (blue) nanodiscs.
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(B) Comparison of cell-type composition between control and EML1-heKO. Numbers indicate percentage of total cells belonging to the respective cell-type. Data information: RG1: radial glia1, RG to bRG: radial glia to basal radial glia, RG2: radial glia to young neurons, IP: intermediate progenitors, YN: young neurons, MLC: mesenchymal-like cells.
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A. Actively proliferating hTERT-RPE1 cells were transfected with scrambled or KV10.1 siRNA, and cell cycle distribution was determined using acridine orange staining. Knockdown of KV10.1 induces an increase in cells with DNA/RNA content compatible with G0 (rectangle). However, the quantitative increase (to approximately 10%) was insufficient to explain the abundance of ciliated cells (over 40%)
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B Table summaries of the microscale thermophoresis binding assay The sequence of the respective synthetic peptides is indicated.
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(B) Separation of IDH-wildtype GBM samples between those displaying high mTOR and microglia enrichment (+ve correlation, group 1 in orange) and those without this signature (group 2 in green).
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D. Comparison of the average cell length of 178 strains obtained from two independent 96-well cultures of the 176 most phenotypically remarkable Keio strains and 2 WT replicates.
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D. Representative P0 superior colliculus stratum griseum superficiale (SGS) (sagittal sectioning of P0 brains along the dashed red line in the schematic, maximal projection of 5 µm depth). Note the presence of Dicer within the SGS marked with neurofilament. n=3 independent experiments. Abbreviations: WT, wild type; P0, postnatal day 0. Scale bars: 30µm.
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B, Venn diagrams illustrating the overlap of genome binding sites between (top) BRPF3, HBO1 and ORC1, and (bottom) BRPF3, BRPF1/2 and ORC1 in human cells.
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G) Gene set enrichment analysis (GSEA) using specific secretory and enterocytic transcriptional signatures with respect to the list of up-regulated genes in Eed-/- crypts.
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B. Representative images of S2 cells expressing GFP fusions (green) of the MDI proteins diagramed in (A). Staining with MitoTracker Red (red) to label mitochondria shows an overlapping signal (yellow), indicating proper localization to mitochondria for all fusion proteins except MDIΔMTS.
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(C) Chromatin states at bidirectionally transcribed predicted enhancers loci. All Bidi loci identified in NBC samples (n = 961) and CLL samples (n = 8530) are shown. The Bidi loci show an enrichment of the states "Active 2 (predicted active enhancer)" and "Active 1 (predicted transcription start sites)"
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GRVillinKO mice were treated with PBS (n=9) or 10 mg/kg DEX (n=10) before 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.
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(D, E) Representative confocal micrographs of control (upper panels), Yip1A-knockdown (middle panels), and IRE1-knockdown (lower panels) cells double-stained for Lamp2 (a marker for late endosomes/lysosomes; green) and B. abortus (red) at 24 hr p.i. BCVs co-localized with Lamp2 are indicated by arrowheads. The infected cells are outlined with white dashed lines. Scale bars are 10 μm. The percentage of Lamp2-positive BCVs was determined, and is shown in the line graph (E).
|
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K. Representative WB of Skm OXPHOS and related system proteins. Two samples per condition are shown. Each sample contains extracts from 3 mice. Histograms represent quantification (wt, n= 6; LowOXPHOS, n= 6). NDUFA9 (CI), SDHA and B (CII), ETF subunits A and B, ETFDH, CoreII (CIII), subunit 1 (CIV), βF1(CV) and hATPIF1 immunoblots are shown. Tubulin is presented as a loading control. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.
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(A) The expression of NLRP3 protein in non treated and LPS/ATP treated wild type and Becn1+/- microglia was determined by Western blot and normalised to ACTIN. Reduction of BECN1 significantly enhanced expression of NLRP3; mean ± SEM, wild type n = 4, wild type LPS/ATP n = 9, Becn1+/- n = 4, Becn1+/- LPS/ATP n = 23; ANOVA with Tukey´s post hoc test *: p < 0.05.
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B GSEA displaying correlations of the gene-expression profiles between control and CDK12-overexpressing cells based on RNA-seq results.
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I, A proposed working model for the p53-USP7-H2Bub1 axis in the regulation of SLC7A11 expression.
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B, H1299 cells treated were subjected to transmission electron microscopy. Representative images are shown. Arrow head indicates condensed and marginated chromatin.
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(F) Immunoblot analysis of the NFATc1 expression level in human keratinocyte HaCaT cells treated with SIRT7 siRNAs.
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Two top-ranked SplashRNA-designed shRNAmirs targeting human Nurd complex components and a control shRNAmir targeting human CD4 were cloned into both miR-AB and miR-E lentiviral vectors with Venus as reporter. The lentiviruses packaged by these constructs were used for transduction of multiple human cell lines. RNAi efficiency was determined by western blot (WB) analysis of target protein levels in the lysates of the transduced cells. -actin served as loading control.
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Relative volume of tumours grown in 12 Rag2-/- animals after treatment with Tepotinib or vehicle control (3K and 3KM tumours for each condition). No statistical significance was found by One-way ANOVA. Mean +/- SEM.
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D. Blood glucose levels for insulin tolerance test (ITT) of control (n=4) or RbpjiΔEC (n=3) mice kept on HFD. Data represent unpaired t-test, mean ± SEM. E. Quantification of AUC for ITT in D. Data represent mean ± SEM, unpaired t-test.
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(A) Representative transmission electron microscope (TEM) image showing the presence of EVs in the CSF in two independent experiments.
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(D) The frequency of contact-dependent assembly (left panel) and number of assemblies per cell per min (right panel) in cells from different strains as indicated on x-axis. Three biological replicates were performed for each strain. At least 500 cells were analyzed per replicate on 3D-SIM time-lapse imaging of sheath dynamics (TssB-sfGFP) in 3 min period. Bar graph (left panel) and scatter dot plot (right panel) show mean ± SD. Unpaired t-test was performed.
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J Quantification of the concentration of DEBIO-0932 reached in animals harboring H2030-BrM established brain metastases 6h after oral administration of DEBIO-0932 at 160 mg/kg. The concentration was measured in both the plasma and the brain for each mouse. Values are shown as mean + s.e.m. (n=3 mice per experimental condition). P value was calculated using two-tailed t-test.
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I) Effect of 24 h incubation with 250 ng/ml TRAIL on Nalm6 199R, Nalm6 199R BAK KO and Nalm6 199R BOK KO, measured as DRAQ7+ cells with respect of total cell surface and normalized to untreated conditions. Each dot corresponds to the average normalized value of 3-4 technical replicates in n=3 independent experiments.
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(A-C) Three different mediation models are shown, testing plasma P-tau217 as a statistical mediator of plaque density on tangle density. Panel A shows the mediation of total tangle density (significant mediation, 60% of the direct effect of plaque density on tangle density was explained by plasma P-tau217). Panel B shows the mediation of medial temporal lobe (MTL, entorhinal cortex and hippocampus) tangle density (no mediation). Panel C shows the mediation of tangle density in all regions except the MTL (strongest mediation, 77%). The P-values are from linear regression models used to estimate the mediation effects.
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Knockdown of Mfn2 inhibited IRE1α ubiquitylation by MITOL. HEK293 cells were transfected with either scramble RNA, siMfn2 and indicated vectors 24 hours prior to immunoprecipitation assay.
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A Immortalized Brca1F/−MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Cells were arrested in mitosis with colcemid and mitotic chromosomes were processed for CO‐FISH analysis. Metaphase chromosome spreads were stained with Cy3‐conjugated leading strand telomeric PNA probe (red) and FITC‐conjugated lagging strand telomeric PNA probe (green). DNA was counter‐stained with DAPI (blue).B The frequency of chromosome‐type telomeric fusions in cells treated as in (A) was quantified as a percentage of total number of chromosomes (illustrated in Supplementary Fig S2A). A minimum of 2,000 chromosomes were scored for each treatment. Error bars represent SD of at least two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; **P ≤ 0.01; NS, P > 0.05.
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B Responses of locust nymphs in a dual-choice test in a Y-tube olfactometer after Piwi1 knockdown (n=46 locusts), ns: not significant (chi-square test). Data information: All assays were performed on fourth-instar locusts.
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A. Representative electron micrographs of HRP uptake from standard (SE) or 100 mM K+ (100K) bath solution. Scale bar, 2 μm.
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B Expression of PDI variants in zebrafish. Zebrafish embryos at 1-2 cell stage were injected with sense mRNA coding for the indicated PDIs (PDIA1WT, PDIA1D292N and PDIA1R300H: 200 pg/embryo; ERp57WT, ERp57D217N and ERp57Q481K: 80 pg/embryo). Protein expression was confirmed by Western blot analysis using anti-V5 antibody in total embryo extracts at 24 h post-fertilization (hpf). Actin was used as a loading control. In the left panel 80 µg protein and in the right panel 120 µg protein extract were used.
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D-E. Respiration of isolated mitochondria from Ifnb+/+ and Ifnb-/- whole brains. OCR measurements under Complex 1 substrates (pyruvate/malate) were obtained at baseline (B) and on addition of ADP, oligomycin, FCCP and rotenone/antimycin A to capture S3, S4o, S3u, and nonmitochondrial respiration. N=3-4 mice per group; bar graphs show mean + SE.
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D) Phylogenetic alignment of the NDP52 zinc finger. Zinc-coordinating residues in bold, conserved residues in the helix in red. The asterisk indicates D439, the residue mutated in subsequent experiments. Hs - Homo sapiens, Bt - Bos taurus, Gg - Gallus gallus and Xe - Xenopus laevis.
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B Biochemical analysis of HOPS intracellular localization. RKO cells were transfected or not with HOPS and fractionated into nuclear and cytoplasmic component. Aliquots relative to the same cell number per fraction were immunoblotted using anti-p53 and anti-HOPS antibodies. GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively. Data information: all the experiments detailed above were performed three times, and representative panels are shown.
|
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G Western blot analysis showing the equivalent expression of the λN-HA-AGO1proteins used in the tethering assays shown in panels (A,B,E,F).
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WT Hoxb8-FL cells were treated with CHIR-124 in the presence or absence of the pan-caspase inhibitor QVD. Cell death was assessed after 24h using flow cytometry as in (A).
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Representative immunoblot of M2 anti-FLAG immunopurification of media conditioned for 24 h on HEK293T cells co-overexpressing FTTTRA25T, ERdj3WT, ERdj3H53Q, and/or BiP, as indicated. Media inputs (1:400) are shown as a control. Lysates from these cells are also shown to confirm the increase of BiP afforded by overexpression.
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F. Representative images (top) and quantification (bottom) of collagen gel-contraction ability of CAFs (monoculture) or CAFs co-cultured with siCNT (co-siCNT) or sihMENA(t) PANC-1 cells (co-sihMENA(t)). Dashed white circles illustrate the margins of the gel area. Data are presented as the mean ± SD of two biological replicates. Statistical analysis was performed with one-way ANOVA P=0.005, followed by Bonferroni's multiple comparison test ANOVA: *P<0.05, **P<0.01.
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B, C Aβ40 and Aβ42 (pmol/g wet brain) in the FA-soluble brain extract from the same APP23mice showed a robust increase with age; ANOVA revealed a significant cubic trend (F(1, 56) = 221.114, P < 0.001 and F(1, 56) = 370.947, P < 0.001, respectively).
|
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