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B-D Strand exchange assay with ssDNA and RAD51, and with (C) or without (B) the addition of MMS22L-TONSL (75 nM).D Quantification shows averages, n = 2; error bars, SEM.
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C) The reductions of treatments compared to IgG control at day 30 did not reach significance. IL-6 levels only very slightly increased from D16 to D30 in the IgG control; while for all other treatment groups there was a trend to reduced IL-6 levels at D30 compared to baseline with the high dose of RG7716 reaching significance, IgG control vs RG7716, 90 μg (*, P = 0.023).
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Representative photomicrographs of cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses, immunolabled for BDNF (green), SCG2 (red) and counterstained with DAPI (blue) in orthogonal views (dashed lines) showing colocalization of BDNF and SCG2 in the soma (white arrows).
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C Images of FOF1 a-mCherry-expressing E. coli fabA(Ts) grown at 30°C or shifted to non-permissive 37°C and stained with Laurdan. For fluorescence intensity correlations, see Appendix Fig S8E.
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MITOL KO did not affect the autophosphorylation of IRE1α. MEFs were exposed with Tu for indicated periods and phosphorylated IRE1α was detected by immunoblotting after Phos-tag SDS-PAGE.
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Average tumor weight of all groups at the end of the experiment described in (A). Error bars indicate standard deviation. Unpaired t-test was used to calculate statistical significance. P-values are indicated in the graph.
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(G) ChIP-qPCR assays was performed for PMAIP1, CDKN1A and SESN2 genes and demonstrated that significantly decreased RNA Poll II enrichment around TSS of PMAIP1, CDKN1A and SESN2 in various stable lncPSCA konckout GC cell lines. Left subpanel: RNA Pol II ChIP-qPCR fragments a, b and c on PMAIP1, CDKN1A and SESN2 genes; middle and right subpanels: RNA Pol II ChIP-qPCR results of PMAIP1, CDKN1A and SESN2 genes in MGC80-3 (middle) and HGC-27 cells (right). Data show one representative example of three biological replicates.
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F. Representative image of tdTomato+ and tdTomato- SCNT embryos retrieved at E6.5. The tdTomato+ SCNT embryo displays normal egg-cylinder morphology. By contrast, the tdTomato- SCNT embryo shows abnormal morphology. Epi, embryonic epiblast; ExEm, extraembryonic ectoderm; EPC, ectoplacental cone. Scale bar, 50 μm
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Western blot analyses in HOP92 cells showing increased expression of collagen I after siRNA against RASSF1A.
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H. Quantification of VAPA signal in TGN46 regions (VAPATGN46). Control: n=44; U18: n=44.
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(D) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells transfected with siRNA targeting PLCβ1 or a control siRNA. For siCntrl, N = 5 mice. For si1PLCβ1, si2PLCβ1, N = 6 mice/group. Right, H&E staining of representative lung sections.
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E. Schematic transition state for the reaction of Ubc1 with K63-linked diubiquitin. Binding of the distal moiety in K63-Ub2 to the UBA domain involves R42 in ubiquitin and enhances targeting of K48 in the proximal moiety for discharge of donor ubiquitin by the UBC domain. This results in the assembly of a K48/K63-branched chain.
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endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (C) HUVEC transfected with control or PECAM-1-specific siRNA were incubated with differentiated HL60 cells for 20 min.
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E. Expression of mavQ in L. pneumophila strains used for infection. Total proteins of bacteria cultured to the post-exponential phase separated by SDS-PAGE were probed with MavQ-specific antibodies. ICDH was detected as a loading control.
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D Linear regression modeling and model selection revealed that while proliferation of mCFU-E cells is best described by integrated pS6 only, for 32D-EpoR and BaF3-EpoR cells proliferation correlates mainly with cell-cycle indicator. Proliferation was measured, integrated pS6 and cell-cycle indicator were simulated with our mathematical model. The linear regression model selection is based on Akaike's information criterion. The respective correlation is given. For details, see Appendix N and O.
|
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(A) Representative whole mount images of WT, Sas4cKO, Sas4cKO;Trp53bp1-/-, Sas4cKO;Usp28cKO animals at P14. Arrows indicate the cerebellar hypoplasia resulting from the lack of primary cilia. Scale bar = 0.2 cm. (B) Telencephalon area of P14 brains of the indicated genotypes. WT littermates N = 8, Sas4cKO N = 7, Sas4cKO;Trp53bp1-/- N = 6, Sas4cKO;Usp28cKO N = 10, Sas4cKO;Trp53-/- N = 5; one-way ANOVA with post-hoc analysis. WT and Sas4cKO data are from Figure 1D and shown alongside for comparison. Black circles represent Nestin-Cre+ animals. (C) Cortical thickness of P14 brains of the indicated genotypes. WT littermates N = 4, Sas4cKO N = 4, Sas4cKO;Trp53bp1-/- N = 4, Sas4cKO;Usp28cKO N = 4, Sas4cKO;Trp53-/- N = 4; one-way ANOVA with post-hoc analysis. WT and Sas4cKO data are from Figure EV1D and shown alongside for comparison. Black circles represent Nestin-Cre+ animals.
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Gel shift assay of biotin-labeled mascRNA with the cytosol in the presence or the absence of a QARS antibody.
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e. Chest radiograph of patient 1 was obtained on day 5 after admission (day 11 after the onset of illness). Bilateral diffuse patchy and fuzzy shadow were observed.
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Localization of all lysine residues (shown as green sticks) on the structure of CtUba4C202K-Urm1. CtUrm1 C-terminus and the lysine residues of CtUba4C202S that become covalently linked to Urm1-COSH in oxidizing conditions are presented in red.
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A Schematic representation of inner ear anatomy, with a focus on the organ of Corti and the cellular function of nesprin-4. B Timeline of experiments performed.
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] |
(B) Expression of BAM3-CITRINE fusion protein under control of the BAM3 promoter in 5-day-old Col-0 roots or makr5 roots.
|
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B. Representative immunoblot of FLAG-tagged REL-OPT and REL-WT in HEK293T cells transfected with either empty plasmids, plasmids bearing REL-OPT or REL-WT. The immunoblot is representative of 3 independent experiments. ACTB is shown as the loading controls.
|
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] |
(G) Multiple color staining was performed in Srcap+/+ and Srcap-/- intestine sections. AP-Red, DAB and AP-Blue were used for immunohistochemistray coloration. Numbers of each kind of epithelial cells from 50 crypts or 50 villi (10 crypts and 10 villi per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bars, 50 μm.
|
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c, The percentage of bacteria in LAMP1+ compartments was quantified and characterized as either spacious or tight-fitting. Mean ± s.e.m. for three mice examined. The image in a (from ref. 2) and the tissue sections were provided by E. Unanue.
|
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B Immunoblot analysis of protein extracts from siCTRL- or siDDR1/2-transfected MM099 short-term cultures plated on FRC- or MAF-derived ECMs in the presence or not of 2 µM BRAFi plus 0.01 µM MEKi for 96 h using antibodies as above. Note that data come from the same immunoblot.
|
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Expression of Rab25 increases endogenous ATP level in ovarian cancer cells. *p < 0.001 Rab25 versus pcDNA. The mean cellular concentration of ATP per mg of protein (based on the standard curve generated using a known amount of ATP) is 2.36−10 moles/mg for IOSE29htpcDNA cells, 6.21−10 moles/mg for IOSE29htRab25 cells, 8.14−11 moles/mg for IOSE80htpcDNA cells, 1.05−10 moles/mg for IOSE80htRab25 cells, 2.85−10 moles/mg for A2780pcDNA cells, 3.81−10 moles/mg for A2780Rab25 cells, 7.23−10 moles/mg for HEYpcDNA cells and 9.82−10 moles/mg for HEYRab25 cells.
|
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] |
A - C Protein lysates were obtained from cortical neuronal cultures treated with 50 mM KCl applied directly to culture media for indicated durations. 100 µm AP5 was used to block NMDAR activity during KCl treatment. Quantification of phosphorylated EB2 (B) and total CDKL5 (C) are normalized to 0 minutes time point. EB2 phosphorylation is significantly decreased after 10 minutes of KCl treatment compared to 0 minutes. CDKL5 protein levels are slightly reduced after 40 minutes compared to 0 minutes. Dunnett's multiple comparison: n = 3 replicates
|
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(A) Scheme showing the FLNA wild-type (wt) allele, the targeting vector and the FLNAΔECS neo-allele. The loxP flanked PGK-neo cassette in the targeting vector replaced the editing complementary sequence (ECS) using homologous recombination, which was then deleted using Cre recombinase. Right, southern blotting analysis screened for positive clones shown. The positions of loxP sites, restriction enzyme (EcoRI) and Southern blotting probe are also indicated.
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F Heatmap of 3D spatial proteomic data showing qualitative analysis of differences in cell type markers and matrix expressions between young and aged murine endocrine glands. The colour code based on immunolabelling intensities indicates very high expression in red, high expression in yellow, medium in light green, low in dark green and no expression in grey.
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(b) RT-PCR analysis of IL-8 and CHOP mRNA expression in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG.
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A, BAverage relative intensities over time and corresponding fluorescence t 1/2 values of heterochromatic Swi6 obtained from FRAP experiments performed with cells expressing NLS‐Swi6‐EGFP (blue) or NLS‐Swi6‐KR25A‐EGFP (red).
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B Cdk5 inhibition by Roscovitine (Ros, 10 µM) rescues the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface induced by VPS34IN1 (10 µM). N = 4 independent experiments (20 images per condition); Two-way ANOVA; Tukey's Multiple Comparisons Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001
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E. The MS/MS spectrum of the peptide (MAAEEKQAQSLQPSSSR) of endogenous PTENε in mice that matches the N-terminal sequence of PTENε. Protein lysates of PtenFLAG knock-in liver tissues were subjected to immunoprecipitation with anti-FLAG M2 agarose. The bound proteins were separated with SDS-PAGE, and gel slices of the band ranked fifth by molecular weight were analyzed by mass spectrometry.
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(D) Representative and summary of immunoblots of lamin B of bone marrow neutrophils from WT and Lmnb1TG mice. Vinculin served as loading control.
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(I) WT (black circles) and Mfn2 KO cells (white circles) were incubated with 1 μM Tg for varying times, and ALIX protein was detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.
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(C) Representative western blots of siControl or siPA200 (2 independent siRNAs, a and b) transfected cells, lysed at indicated time points after UV-irradiation (16 J/m2)
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Kaplan Meier survival curve of N2, odr-3(n2150) and odr-3(n2046) worms on partial lawn of P. aeruginosa at 25°C. Kaplan Meier survival curve of N2, odr-3(n2150) and odr-3(n2046) worms on full lawn of P. aeruginosa at 25°C.
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(H) Correlation between LSD1 and SOX2 in 172 gastric cancer tissues (Pearson's test).
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(A-D) ChAPs are differently dependent on each other in terms of binding at the TGN. Binding kinetics of GFP-tagged Chs6 (A), Bud7 (B), Bch2 (C) and Bch1 (D) at the TGN in the presence (WT) or absence of the other ChAPs (). The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.
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(e) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP-Cx43 but knocked down for Eps15. Single channels are shown in reverse black and white, and merged images in colour. Inset shows higher magnification. Bottom: Quantification of co-localization between Atg16 and Cx43 (n = 3 wells, 4 independent experiments, >20 cells per experiment). Values are mean + s.e.m. * P 0.05.
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Binding of SARS nAbs to SARS-CoV S1 protein were tested by ELISA. Recombinant S1 protein of SARS-CoV were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein.B. Binding of SARS nAbs to SARS-CoV-2 S1 protein were tested by ELSIA. Recombinant S1 protein of SARS-CoV-2 were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein.
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HEK293T cells transiently transfected with Flag-PARP1 and Myc-BRD7 for 24 h were lysed with RIPA buffer. Followed by immunoprecipitation (IP) using antibodies to Myc (C) conjugated to agarose followed by Western blot with the indicated antibodies (n=3).
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C Unsupervised hierarchical clustering of the 500 most variable genes across samples. Normalized expression levels were scaled to Z-scores for each gene.
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(E) Representative confocal images (top) and quantification (bottom) of Oct4-GFP reporter mean intensity. The indicated cell lines were treated with DOX for the first 2 days and imaged at Day 6. Mean +/- SD of 9 technical replicates. One representative experiment of two is shown. Scale bars=300µm
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(B) Western blot analysis of LAMP-1, a late endosome marker, which was nearly identically distributed over the first two fractions compared to Cathepsin-D, in the controle group, but ocurred only in the first fraction in the OMP treated group. β-COP as an indicator of the Golgi-complex, is found strongly in the lower fraction in the control group, but very weakly in the OMP treated group. Cathepsin - D, an early endosome marker, which can be found in the upper and middle fraction of the control group, but regarding the OMP group, it was only found in the first one.
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Expression of CBFs in EGR2-overexpressing plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis. Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).
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MSN cultures were stimulated with HiK (55 mM) for 1h in the presence of various pharmacologicals to dissect the synapse-to-nucleus cascades mediating transcription of Npas4, Fosb and Arc. Values were normalized to the induced condition to compare for differences in induction between treatments. Mean + SEM and one-way ANOVA with Bonferroni post-hoc correction were used in all experiments. p* < 0.05, p** < 0.01, p***<0.001, p****<0.0001. n-number indicated in each panel. g, blockade of CaMKII/IV through application of KN-93 (1 µM, 20min before) leads to significantly reduced mRNA inductions of Arc, Fosb and Npas4. Mean + SEM; n = 4 independent cultures as biological replicates. h, co-application of HiK and SKF reveals synergistic effects on mRNA induction of Fosb but not Arc or Npas4, Mean + SEM; n = 6 independent cultures as biological replicates.
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miR-AB cloning efficiency. Various amount of BamHI/ApaI-cut miR-AB vectors were ligated with miR-AB oligos (keep the ratio of vector mass to oligo mole at 1:1; for example, 1 ng of vector with 1 nmol of oligo) and colonies were counted after transformation of XL-10 Gold competent cells (homemade, 2 X 109 transformation efficiency). The four colored lines represent four individual shRNAmir cloning efficiency from two independent experiments (two individual shRNAmir cloning in each experiment).
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Representative pictures (left panel) and the normalised biosensor intensity based on live cell imaging of the cells with a GSK3-GFP biosensor following different treatments before and after metaphase (n=10). Scale bars represent 10 μm.
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(A) Overview of the multi-omics data integration workflow followed in this study. Proteomic and transcriptomic data generated as part of this study or available from the literature for colorectal cancer (CRC) cell lines and patients were integrated in order to identify cell lines and tumours forming proteomic subtypes. Four published drug sensitivity datasets (abbreviated CCLE, CTRP, GDSC and Cetuximab (Medico et al.); one dose response plot for each data source in shown) were overlaid onto the proteomic data to identify protein signatures associated with sensitivity or resistance. An example of an effect-size heat map for one drug, ten proteins and 20 cell lines is shown at the bottom-right (see main text and Appendix for details).
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(A). Cell growth assessment of ΔIndLon (upper plot) and ΔIndFtsH (lower plot) mutants grown under inducing (blue) or depleting conditions (red). Growth was monitored by measuring DNA and protein biomass over time. The average from two independent biological replicates is shown.
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PRKRIR, PCBP4 and TSC22D1 are crucial for maintenance of the mesenchymal fate. (A) Scratch assay in MDA-MB-231 cells treated for four days with DMSO or JNKi. Scale bar, 200 μm; 20 X magnification. Mean and SEM is plotted from three independent biological replicates.
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(a) NRK cell lines stably expressing CFP-LC3; LAMP1-YFP were transfected with nonspecific (NS)- or PIP5K1B-RNAi and starved for 12 h. Scale bars, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.
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(C) Fluorescence intensity based analysis of pFTAA-stained Aβ plaques in the cortex of male (n=10) and female (n=8) APP23 mice (left) and representative images (right), scale bar = 1 mm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, **p=0.0011.
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(A) Schematic of the σV circuit. In the figure R (orange) is the anti-sigma factor RsiV, R* (orange) is RsiV bound to lysozyme, S (red) is signalling peptidase and RP (light blue) is RasP the site-2 protease. For more information on the activation mechanism see Figure 1B.
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SDS-PAGE of Tau incubated in LLPS (PEG, tRNALLPS, PEG:tRNALLPS) or aggregation (hepAGG) conditions for 1h, 24h, or 72h. SDS-stable Tau dimers and oligomers species evolve in LLPS and hepAGG samples after 24h. After 72h, all conditions contain SDS-stable Tau oligomers and dimers.
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D. Hippocampal neurons were cultured in a multi electrode array (MEA) plate equipped with sixteen electrodes. Spontaneous activity of the neurons was recorded at every 3 hours (10 minutes/session) for 24 hours. Weighted mean firing rate, number of bursts, and network bursts are significantly decreased in neurons treated with 3-miR-mix compared to control.
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(B) Schematic representation of the predicted m152 (red) and STING (grey) topology in the ER membrane. (1) and (2) specify the ER luminal loop regions of STING with the sequence alignments from murine and human STING shown below. In murine STING, N41 in loop (1) was mutated to E41 and loop (2) was exchanged with loop (2) of human STING (hL2). The resultant constructs were designated mSTING N41E, mSTING hL2 and mSTING N41E-hL2. In human STING, mutations were introduced vice versa, resulting in hSTING E41N, hSTING mL2 and hSTING E41N-mL2.
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(G and H) IEM followed by silver stain enhancement for Aβ40 was performed in 9-mo-old PS1/APP mice.
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C. Centrosome (PCNT) separation was assessed in Aurka; Plk1 dKO spermatocytes. Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation classifications
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E. Percentages of cells with indicated mitochondrial morphologies in WT and Drp1-/- 293T cells transfected with empty vector (control), Myc-hFis1 and either Myc- or GFP-tagged mutants as indicated in three independent experiments.
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(A)-(D) Expression of (A) HNRNPA0, (B) U2AF1, (C) SF3B3, or (D) NECAP1 72 hours post-transfection with either SMN2 SSO or knockdown of each target using siRNAs in HEK293T cells. (E)-(G) Expression of (E) SMN2-FL, (F) SMN2∆7, or (G) SMN1-FL following SMN2 SSO or siRNA transfection (n=4). Data information: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; empirical p-values are determined by a simulation and sampling method The error bars show the Standard Error of Mean (SEM). Data from A-G represent 4 biological replicates. Three technical replicates were performed during qPCR for each biological replicate and averaged. Scrambled siRNA condition for each experiment was set to 1.
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A Immunofluorescence staining of HK2 with an AlexaFluor488-conjugated antibody in HeLa cells expressing mitochondria-targeted RFP. Yellow signals in the merge analysis indicate mitochondrial localization of HK2. Scale bar: 15 µm.
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Mouse livers treated with vehicle or 1 μM rapamycin (Rapa.) for 16 h by an intraperitoneal injection were fractionated into cytosol (Cyt.) or mitochondrial (Mito.) extracts and analyzed by western blotting. Phosphorylated (S2468) mTOR (p-mTOR) was used to assess the rapamycin activity. E-cadherin: plasma membrane marker; β-tubulin: cytosolic marker; VDAC: mitochondrial marker; Homo: cell homogenate
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H, I Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=6; *: p<0.05 and **: p<0.01 by t test
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Kaplan-Meier survival curves of nude mice transplanted with MDA-MB-231 cells and injected with CIP2A-BP or svCIP2A-BP (10 mice per group). The horizontal line indicates the time after the mice were injected with the cells via the tail vein.
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Two-photon imaging of these mice confirmed that astrocytic hyperactivity were reduced by MRS2179 (APP/PS1-Stat3WT, n = 6 (4 female and 2 male) mice; APP/PS1-Stat3KO, n = 6 (2 female and 4 male) mice; age, 8 months; Mann-Whitney test).
|
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A. Relative antiviral activity of IFNα (circles) or IFNλ (triangles). AEC cultures were stimulated for 4hrs with stated IFN at specified concentrations (ng/ml) and induction of indicated ISGs was assessed by qPCR (data shown is representative of four independent experiments, n=3-4).
|
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E αSMA mRNA expression in TGFβ-treated IRE1α -/- MEF cells that were either untransfected (UT) or transfected with miRNA inhibitor control (siCtrl) or with a miR150-5p miRNA inhibitor. *P = 0.043 compared to UT.
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(B) Total glycogen in the indicated tissues, collected from n=5 nine month old vehicle and 144DG11-treated mice, was quantified Repeated-measures 2-way ANOVA tests show that the pharmacokinetic profile of each tissue is significantly different from that of all other tissues (p<0.05). *, Significant difference (p<0.05) as determined by two-tailed t-tests.
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F) Microscopy of BMDMs (Zdhhc18+/+ or Zdhhc18-/-) infected with GFP-HSV-1 (MOI=1) for 10 h. Scale bar: 200 μm. Data information: Data are representative of at least two independent experiments.
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(B) Immunoprecipitated HA-tagged C9ORF72, HA-tagged SMCR8 and HA-tagged WDR41 expressed in HEK293 were subjected to in vitro TBK1 kinase assay in the presence of γP32-radiolabelled ATP. Proteins were separated by SDS page migration and phosphorylation was detected by autoradiography (upper panel), while expression was detected by western blotting (lower panel).
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E Cr-release assay showing % specific lysis of MDA-MB-231 breast tumor cell line by survivin-specific T cells at different ratios upon CCR9 knockdown (○) in comparison to the control knockdown (▪).
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(a) Midguts dissected from animals expressing Atg18IR specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast (DIC) microscopy. Representative images are shown. (b) Quantification (μm2) from a, n = 12 animal intestines per genotype with 1-5 cells measured per intestine.
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C. Mapping of flam-piRNAs produced in the presence and absence of the Hel-C domain in OSCs. D. Mapping of tj-piRNAs produced in the presence and absence of the Hel-C domain in OSCs.
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F. siRNA mediated knockdown of VEAL2 significantly reduces expression of VEAL2 in HUVECs. Bar graph representing relative expression of VEAL2 in control siRNA and VEAL2 targeting siRNA transfected HUVECs. Data is acquired from 3 different biological replicates and shown as mean fold change values ± standard deviation.
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(b) GST-RILP pull-down analysis from HeLa cells in normal and starved conditions showing an enhanced capacity of endogenous Rab34 to bind RILP in starvation conditions. Quantification is mean of 3 independent experiments. Error is S.E.M.
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(G) Breakdown of mapped reads at the annotated genomic regions. Gene body: 3'-UTR, exon, intron, 5'-UTR; Others: ncRNA, miRNA, snoRNA, and pseudogenes. The proportions of the annotated region on the mouse genome are shown as "Genome" at the bottom lane.
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Lysates from BT549 transfected with HA-FGFR1c, 2b, or 4 and stimulated for 8 or 40 min. with Enkamin-E, FGF10, or FGF1, respectively, followed by treatment with either DMSO or the FGFR inhibitor PD173074 (B)
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B Frequency distribution of nucleosomal DNA size (WT in grey fill with nap1Δ in blue traces) from H3 ChIP-seq.
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RNAi was induced with an empty vector as a negative control (panel I), whereas a smg-2 clone (panel II) was used as a positive control. Panels i and ii show brightfield images of the PTCxi strain treated with the negative and positive controls, respectively. Depletion of five genes (panels III to VII) resulted in increased GFP expression. Panels iii to vii show brightfield images of the phenotypes of the affected worms. The scale bars correspond to 100 μm.
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(H) Number of SQST-1 aggregates per focal plane in wild type, atg-3 mutants, and epg-7 mutants.
|
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(A) 293/GFP-LC3 cells were transfected with control or p38α siRNA. At 72 h after transfection, cells were incubated in either full medium, EBSS, or EBSS with leupeptin for 2 h, then fixed and visualized by confocal microscopy. Bars=5 μm (data are represented as mean±s.e.m. n=60 cells, EBSS control versus p38α siRNA (***P=0.0001); EBSS with leupeptin control versus p38α siRNA (***P=0.0001), Student's t‐test).
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Northern blotting of mascRNA and 5S rRNA in the lysates of human embryonic stem cells on different days of differentiation.
|
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B Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in lysates of TSC1-KO MEFs transfected with (+) or without (-) TSC1 wildtype or K30A expression vectors. The cells were serum starved and then left either untreated (NT) or stimulated with 10% FBS for 30 mins. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC2 (right panel) protein bands relative to the level of β-Actin.
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(B) Macrophages transduced with shNS or shTLR8 from (A) were exposed to infectious HIV (+), AT-2-inactivated HIV (AT), or RNase/DNase I treated AT-2-inactivated HIV (R) or mock infected (-) for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.
|
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C. Cells were treated with BMP2 for 1 h. D. Cells were stimulated with increasing (doubling) doses of BMP4/7 (0.625-20 ng/mL) for 1 h. Data information: In all cases, whole cell extracts were Western blotted using the indicated antibodies. Representative experiments are shown. The levels of pSMAD1/5 relative to Actin were determined and are expressed as a fold change relative to the parental HEK293T untreated sample or normalized to the 20 ng/mL ligand dose in parental HEK293T cells Act, Activin A; Par, parental HEK293T cells, KO1, ACVR1 KO clone 1.
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The interactions between YTHDF1 and ZAP (B) were measured by co-IP in 293T cells. The results were detected using specific antibodies.
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(C) Mean expression levels (RT-qPCR) in white vastus (WV) and soleus (Sol) muscle from 8-week-old male wild type mice (n = 5 mice per group). *p < 0.0001 (Myh7b), *p < 0.0001 (miR-499), *p < 0.0001 (Myh7), *p < 0.0001 (miR-208b).
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Quantification of Kcnq1-positive marginal cells and the effect of treatment on the cellular organization of the marginal cells in the SVThe membranes of marginal cells are labeled (red) by phalloidin conjugated with rhodamine.A Immunolabeling results (Kcnq1 labeled in green) in WT mice.B, C Immunolabeling results (Kcnq1 labeled in green) of treated Kcnq1−/−mice, middle (B) and apical (C) turns, respectively.D The percentage of marginal cells having positive Kcnq1immunolabeling signal above a visually detectable level is shown for WT (gray bars, left), untreated Kcnq1−/− (middle), and treated Kcnq1−/−mice (black bars, right). Data are given as mean ± SD (n = 6).E, F The organization of marginal cells in the SV is outlined by labeling with phalloidin conjugated with rhodamine. Results from WT (E) and untreated Kcnq1−/−mice (F) are compared.Data information: Scale bars represent approximately 50 μm.
|
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Representative flow cytometric dot plots and histograms of PDGFRα expression in rMC (EpCAM-CD45-CD31-Sca-1+) isolated from the lung homogenate of PdgfraGFP reporter mice.
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A. Dimensionality reduction using UMAP. Each dot represents a cell. Colors indicate AC and DMZ.
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A. EYFP-Parkin translocation requires Ca2+. PC6 cells plated in separated compartments of the same dish maintained during the experiment in Krebs containing 1 mM Ca2+ or in Ca2+ free Krebs containing 5 μM BAPTA-AM, were treated with A&O (both 10 μM). Images (left panels) demonstrate that EYFP-Parkin translocation was significantly weaker at zero Ca2+ at 195 min after A&O treatment (compared with 15 min; note the spatial heterogeneity values shown in photos). Spatial heterogeneity analysis (right panel) demonstrated that Parkin translocation was significantly decelerated in zero Ca2+ conditions (n = 25-27 cells; difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)
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(c) 2D-[15N, 1H]-TROSY spectrum of BakΔTM in nanodiscs after Bid-BH3 activation with the assigned resonances indicated.
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A-C Thiophosphorylation assays of wild type and mutated forms ARR1/10/12 by MPK3/6 in vitro. Potential phosphorylated Ser residues or Thr residues of ARR1, ARR10, and ARR12 were mutated to Ala (ARR1m/ARR10m/ARR12m). Recombinant ARR1/10/12-His and ARR1m/10m/12m-His proteins were incubated with MPK3 and MPK6 which were activated by MKK5DD. Phosphorylated ARR1/10/12, ARR1m/10m/12m, and MPK3/6 were visualized by an anti-thiophosphate ester-specific antibody (α-TPE) (upper panel). Asterisk in (B) indicates non-specific bands. Recombinant MKK5DD, MPK3, MPK6, ARR1/10/12, and ARR1m/10m/12m were detected by comassie brilliant blue (CBB) staining as loading controls (bottom panel).
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Snf1 maintains glycolysis and glucose transporter gene expression in both WT and ρ0 cells, and represses ribosome and Ribi gene expression in ρ0 cells. ρ0 snf1∆ cells were prepared by acute EtBr (25 µg/ml) treatment of snf1∆ cells in SCD for 2 days. RNAseq was performed with three biological replicates per genotype (Dataset EV8). All the transcripts and two categories of transcripts (gene lists in Table EV1B) are shown. Significantly changed genes (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed genes are indicated.
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E. Representative western blot of O-GlcNAcylation and pro-caspase-3 cleavage in THP-1 cells treated with Stx2a (10 ng/mL) in the presence or absence of OGT inhibitor OSMI-1 (10 µM final). F. Quantification of the band intensities for O-GlcNAcylation (RL-2) in (E). Data are presented as mean ± S.E.M. (n = 3 biological replicates) normalized against β-actin, which was used as a loading control. The effects of Stx2a-mediated induction for O-GlcNAc levels at each time-point were compared to 0 h (left panel), and OSMI-1 treatment was compared with that of the vehicle (DMSO) control at each time-point (right panel). Data information: Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.
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WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h. Snail and Slug was assessed by immunofluorescence staining (D) Scale bar: 20 μm. n = 3 independent experiments, n = 18 fields analysed.
|
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F) Cells were infected with HIV-SARS-2. Luciferase readings at 48 h post infection were normalized to vector control, which was set to a value of 100. Bars represent averages with individual data points from 3-7 independent experiments shown as circles. Error bars represent SD. #p<0.05 by ANOVA followed by Tukey's multiple comparisons test for the comparisons indicated by lines.
|
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(A) (left) Representative images of anti-GFAP immunostaining in Prefrontal Cortex and Amygdala from AppNL-G-F mice receiving either AAV-VHH-B9 or AAV-GFP; (right) quantification of the GFAP+ cell number in the indicated brain regions across experimental cohorts.
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