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A) Epifluorescence microscopy images of MoDCs infected with pre-labeled bacteria (green-CFSE). LAMP1 detected by transduction of red fluorescent protein (RFP) chimera using baculovirus transgenes to MoDCs.
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(c) MN-1 cells were immunoblotted for LC3 in the presence or absence of ammonium chloride to evaluate autophagic flux. A representative LC3 western blot is shown. All ratios were normalized to MN-1 WT cells at baseline, which was set to 1. (d) The ratio of LC3-II:actin for c determined by densitometry analysis using ImageJ. n = 3 independent experiments, F = 0.4501, one-way ANOVA with post hoc Tukey test. *P 0.05.
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(C) The 38 upregulated genes identified (>2 fold where padj=<0.05) were converted to DAVID IDs and used for gene ontology analysis. 3 gene clusters were significantly enriched (p-value = <0.05, drawn on the plot as a dotted line) in the data set: zinc finger proteins (p=2.2 x 10-7), negative regulators of metabolic processes (p=0.0036) and angiogenesis (p=0.011). Venn diagrams on the right show numbers of upregulated genes, ZNFs and KZNFs and the overlap.
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Liquid chromatography-mass spectrometry (LC-MS/MS) analysis was performed to determine different metabolites of RK-33.
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Kaplan-Meier analysis of tumor formation in control (blue, n = 8) and GS493‐pretreated (red, n = 8) MLN4924;MMTV‐Cre;Shp2fl/flmice. The bar indicates the duration of treatment.
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(B) LC-MS/MS spectra of the N-terminal part of the GFP-immunoprecipitated and LysC digested polyGA protein expressed as in (A).
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(a) Levels of dark, dronc, drice and rpr transcripts were measured from third instar larvae (L3), 6 h and 14 h RPF salivary glands from control (blue) and dUTX1 (red) by qPCR and expressed relative to the internal control gene rp49.
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(C) Comparisons of the flap domain of PPM1H with PPM1A (left) and the tudor domain (right). Apart from a conserved loop (dotted circle) which forms an interface with the catalytic domain, the sequences and structures of flaps are diverse among the PPM family.
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Incubation of human melanoma with human platelets or ASM for 5 min triggers co-clustering of ceramide and activated β1 integrin on the surface of the tumor cells. Cells were stained with FITC-coupled anti-active β1 integrin (HUTS-4) antibodies and Cy3-coupled anti-ceramide antibodies. Shown are representative stainings from each four independent experiments.
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B. Red and green pixels overlap fraction (above, left) representing the co-localization of FGFR2 with EGFR; red and far red pixels overlap fraction (above, right) representing the co-localization of FGFR2 with the recycling marker Tf; far red and green pixels overlap fraction (below, left) representing the co-localization of EGFR with the recycling marker Tf; green, red and far red pixels overlap fraction (below, right) representing the co-localization of FGFR2, EGFR, and the recycling marker Tf in T47D stimulated for 40 min. Values represent the median ± SD of at least 3 independent experiments. Representative pictures are shown in A and Appendix Figure S6A. *, p value<0.005 (Student's t-test).
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(U) Representative image of Western blotting analysis of HIF1α, REDD1, 4E-BP1, S6 and P70S6K (in their phosphorylated and unphosphorilated form) to test mTOR activation in normoxic (NRX) and hypoxic (HYP) IL10 macrophages treated with glufosinate (20µM), rapamycin (20nM) and a combination of both (n=3).
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Activity profiles (Tx) of regulatory sequences from donor phyla Proteobacteria and Firmicutes in 10 bacterial species. GC contents of regulatory sequences from the phylogenetic groups are shown on right. Box plots are displaying the interquartile range (IQR) with median values (black line) and whiskers extending to the highest and lowest points within 1.5x the IQR. For normalization, transcription levels in log10 scale were transformed to Z-scores. All measurements are based on two biological replicates.
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I-K Anti-miR-210 delivery (N = 10/group) ameliorated the elevation of RVSP, **P = 0.0031 (i) and vascular remodeling, as reflected by increased percent of muscularized arterioles (N = 8/group, **P = 0.0091) (J) and increased medial thickness relative to vessel diameter in < 100-μm pulmonary vessels (N = 6/group, **P = 0.0021) (K).
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(B) Heavy/light ratio for approx. 500 proteins identified in the screen. SPATA2 stands out with a substantially elevated H/L ratio. Each dot represents a protein.
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b, Percentages of autophagic vacuoles (AVs) containing only lipid, other cargo or mixed cargo calculated from samples processed as in Supplementary Fig. 16b (*P 0.001, **P 0.0001, n = 46). (Supplementary Figure 16 Effects of starvation on hepatic lipid droplets. a, Mouseliverelectron micrographs. Arrows: Lipid-containing double membrane vesicles. b, Electron micrographs of autophagic vacuoles (AVs) isolated from mice fed or starved for the indicated time periods. The percentages of double membrane vesicles containing lipid in isolated AVs from 3-4 mice per condition are shown (*P<0.01 as compared to fractions from fed animals). (*) indicate lipid)
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(A) Proposed models for repair of DSBs generated at broken replication forks (see text for details). seDSB: single-ended DSB; deDSB: double-ended DSB. Pink arrows: endonucleases to remove Flex1 or other secondary structures at DSB ends or DNA tails at the fork junctions.
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(B) A summary visualization of the metabolic pathway enrichment result for the top consistent DF reactions across the datasets, with more importance given to the various in vivo patient datasets (Methods). Y-axis represents the odds ratio of enrichment, the dot color corresponds to the adjusted P value from Fisher's exact tests, and dot size corresponds to the number of enriched reactions within each pathway. Half-dots plotted on the top border line correspond to infinity odds ratio values. The pathways on the X-axis are ordered by P value and only those with FDR<0.1 are shown.
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(A) Mass spectroscopy analysis of levels of cholesterol and cholesteryl esters in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age (mean±SEM, n=10, 4 and 5 mice per genotype respectively; COH=cholesterol, CE=cholesteryl ester).
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A Schematic of the control of lipid synthesis by estradiol-inducible expression of ino2*.
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Drawing of the TSC1 promoter and luciferase-reporter constructs with the predicted E-box (CACGTG, pos. -317/-322) indicated. Relative luciferase units (RLU) of co-transfection experiments using the indicated TSC1-promoter-reporters with MYC normalized to empty pcDNA3 expression vector in HeLa cells (mean ± st. dev., n=3). Immunoblot shows MYC overexpression and α-tubulin as loading control.
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(D) Gastrocnemius weight in mice subjected to iron deprivation by feeding with an iron deficient diet (IDD) combined to a phlebotomy (PHL) (n=5-6); Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
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Figure 4. Deletion of both β-catenin and Hoxa9 abrogates leukemia development in LSK-MLL-ENL cells. Keys and colour code in the left top corner indicate the origin and the β-catenin and Hoxa9 status of MLL-ENL transduced cells. (A) Colony numbers in serial replating assay of MLL-ENL transduced cells. Images of typical 4th round colonies shown above. Data are represented as mean ± SD.
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(C) Scheme of ketone body oxidation and the tricarboxylic acid (TCA) cycle. MCEC were incubated with 1 mM 13C4-β-hydroxybutyrate or 1 mM 13C4-acetoacetate for 24 hours or the same concentration of unlabeled ketone bodies ([12C] isotope). The [13C]-labeled fractions of the TCA cycle intermediates citrate, α-ketoglutarate and malate (marked with blue star) were quantified with an untargeted metabolomics approach indicating that 13C carbon from ketone bodies was incorporated into TCA cycle intermediates.
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F Images displaying the collagen (blue) revealed by Masson's trichrome staining in limb of WT, mdx, and mdx/CD38-/- mice. The dot plot shows the quantification of collagen staining area (% total area) in limb of WT (n=4), mdx (n=5) and mdx/CD38-/- (n=5) mice. Scale bars: 200 µm. Data information: Each dot of the graphs represents a mouse. in triplicate. After normality and variance comparison tests, significance was assessed using: : ANOVA followed by Fisher's LSD test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.
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B. Deconvolved fluorescence micrographs of Chm7-GFP and truncations with Nup170-mCherry NE marker and merge of green and red channels. Scale bar is 5 μm.
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A Reactome Pathway analysis for all 174 transcription factor genes with higher expression in DIE (upper) and all 205 genes higher in CTX GM (lower) astrocytes.
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B HeLa cells were fixed and labelled with antibody against SEPT7 and secondary antibody coupled to HRP, and processed for TEM. The right image is enlarged from the boxed region in the TEM image, and shows a SEPT7 (S7) filament (labelled in black) intersecting with the mitochondrial (Mt) fission site. The scale bar represents 100 nm. See also Fig EV4.
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I, Response of fliM∆N ∆cheA cells expressing FliMN-CheY (strain EW696) to acetate and benzoate (10 mM each; pH 7.0). FliMN-CheY expression was induced with 800 μM IPTG. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The black arrow indicates the estimated time point at which the stimulus entered the flow chamber.
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Morphological evaluation of IMCs cultured for 2 weeks without additional cytokines. The cells grown on culture plates were imaged by phase-contrast microscopy (left). Then, the cells were trypsinised and smeared on glass slides for Giemsa staining (right). These cells are intermediate to large sized, round or amoeboid cells with cytoplasmic vacuolation, consistent with a macrophage morphology. Scale bars, 50 μm.
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K. Relative expression of veal2 across Fluorescence activated cell sorted (FACS) GFP(+) endothelial cells (EC) and GFP(-) non endothelial cells (NEC). fli1a and actb were taken as positive control and normalization control, respectively. Data from three different experiments represented as fold change relative to EC values ± standard deviation.
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Quantitative PCR (qPCR) analysis of YAP/TAZ targets (Cyr61, Thbs1, Amotl2), ATF4 and the targets of ATF4 (Trib3, Slc7a5, Slc7a11).
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(A) Workflow of the experiment presented in panel (C), depicting synaptoneurosomes (SN) isolation and in vitro stimulation in the presence of radioactive 35S-labelled methionine/cysteine mix.
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A Schematic depiction of the growth assays. Cells expressing only one hexose transporter at a time were picked from a plate, serially diluted and plated on fresh YPD plates.
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(F) Representative immunofluorescence images of Spindly phS499 levels at unaligned and aligned KTs in a PoloWT-EGFP expressing Drosophila S2 cell. Insets show magnifications of the outlined regions, which highlight either a unaligned (u) KT or an aligned (a) KT. CID was used as a KT reference. Plotted profiles of signal intensities of phS499 and CID are shown for the highlighted KTs. Data information: Data are shown as mean ± SD. Scale bar: 5 μm.
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c. Statistical analysis of the frequency of IFN+Foxp3+ cells in Tregs stimulated as in c of n=8 experiments performed.
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H) (top) 4 months old and 15 months old micelivers [control (-) or rapamycin (+)] stained with Sen--β-Gal solution (Sen--β-Gal activity is evidenced by blue staining). Data are from n=3 mice per group; (bottom) Representative image showing Sen--β-Gal staining (Sen--β-Gal activity is evidenced by blue staining) in hepatocytes and corresponding immuno-TeloFISH (arrows represent co-localizing foci); Asterisks denote statistical significant P<0.05 using One-way ANOVA.
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A) Survival of P. gingivalis strains within MoDCs after 6, 12, 24 and 48 hours. Blue lines show P. gingivalis strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines.
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C. The five contacting interfaces between MvcA(green surface) and ubiquitin(magenta), each is indicated by a dashed circle and a letter from A-E. The image on the right is obtained by a 180° rotation of the left structure around the indicated axis. The interaction patch involving the C-terminal tail of ubiquitin is indicated by dashed circle. CTC: carboxyl terminus contacts.
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(a) Widefield immunofluorescence images of PFA fixed HeLa cells showing endogenous FLCN and LAMP1 staining in (top) normal growth or (bottom) starvation conditions. Orange arrows highlight association of FLCN with LAMP1 positive membranes in the peri-nuclear region. White dotted line shows cell periphery in starved condition. Scale bar = 10μm.(b) (left) Quantification of FLCN/LAMP1 association in growth and starvation conditions. A cell is scored as positive if 5 or more discrete FLCN/LAMP1 puncta were observed at 100x magnification using a widefield microscope. Data represents 60 cells from 3 independent experiments, error bars show S.E.M, *** = p<0.001. (right) Western blot shows levels of phosphorylated of S6K and 4EBP in whole cell extracts under the same conditions.
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(B-F) Suppression of X-ray-induced autophagy in PPM1D−/−thymocytes. (D) The indicated thymocytes were X-ray irradiated (5 Gy), and 3 hr later, thymocytes were subjected to EM analysis. An autophagic vacuole (arrowhead) can be seen in an irradiated PPM1D+/+thymocyte.
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G, Fluorescent in situ hybridization for cd74a, tnfa, and ctsc at 7 dpi, respectively. mpeg1 was used to label all MC cells. The blue boxed region is highlighted in the zoom-in image to the right. Scale bar = 50 μm. A stands for arium, V stand for ventricle and OFT stands for outflow tract. White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane.
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(A, B) Production of cytokines TNF-α, IL-1, IL-6, CXCL15 (A), or chemokines Ccl5, Cxcl10, Icam1 (B) in the A20-/-Socs6+/+, A20+/+Socs6+/+, A20-/-Socs6-/- and A20+/+Socs6-/- BMDMs after Mtb stimulation. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.
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(A Representative images of HUVECs (A; n=6) sprouting in a fibrin gel with VEGF (25ng/ml) in the presence or absence of COCO. (D Quantification of tube surface area of micrographs shown in (A, (D: *P=0.0119 (- vs COCO 30ng/ml); *P=0.0108 (- vs COCO 60ng/ml)
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Bar graph comparing the number of the cell:cell adhesion proteins identified in each protein interaction dataset. All scale bars 10μm. All error bars represent s.e.m.
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C. Representative ATPase activity assay (ATP regenerating system). At the indicated time, 200 nM ConA is added to the assay.
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(E) Immunoblots of cell lysates to analyze phosphorylated TAK1 by mouse peritoneal macrophages isolated from WT or Galectin-9 KO mice stimulated with AG (1 μg/ml) for indicated times; GADPH of cell lysates served as the loading control. Data are representative of at least n=3 independent experiments.
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(E, F) Exercise capacity of MCK-KO and WT mice from the high intensity regiment (n = 5 mice, means ± SEM, two-tailed student's t-test).
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A panel of selected breast cancer and near normal cell lines was reverse-transfected with 5 nM pooled CEP55 siRNAs and cell viability determined after 6 days. Cell viability relative to its own respective control transfected with scramble siRNA was calculated. Graph represents the mean± SEM of three independent experiments.
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B-G. intracellular ATP levels (E-G) were measured in N2a-IP3R3 cells expressing Sig1R-FLAG (B and E), SOD1 (C and F), or both (D and G). Mean ± SEM from three independent experiments is plotted. *: p < 0.05.
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A. PCR analysis of the human H49K variant of the ATPIF1 and rtTA constructs in wild-type (wt), ACTA1-rtTA (T), ATPIF1H49K-TRE (H) or double transgenic (T/H) mice.
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A. Numbers of ATAC peaks called separately in ESC+2i and ESC+serum including their localization according to compartmentalization. B. Numbers of significant changed ATAC peaks in ESC+2i vs. ESC+serum and their localization according to compartmentalization. Increased chromatin accessibility sites (IAS), decreased accessibility sites (DAS). C. Average profile of normalized ATAC read counts over differential peaks in ESC+2i and ESC+serum changed ATAC peaks. The signal is plotted over +/- 1kb on the center of the ATAC peak.
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E. Rabl2Q80L retained ciliary GPR161 after SAG stimulation. RPE1 cells infected as in (B) were serum-starved for 24 h, followed by DMSO (mock) or SAG treatment for 24 h. Arrows indicate cilia that are displayed in insets. At least 100 cilia were scored in each experiment and condition.
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(B) Depiction of the Vav1 mutants used in these experiments. The E201A mutation is shown as a gray circle. The activity of each mutant is represented on the right. Green box, WT activity; red box, gain-of-function; blue box, loss-of-function.
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A-C. Consequences of effector depletion on the endogenous Arl8b/Rab7 hybrid compartment. A. Representative confocal images of fixed HeLa cells harbouring GFP-tagged endogenous Arl8b (G-eArl8b, green), transfected with the indicated siRNAs and immunolabeled against endogenous Rab7 (eRab7, magenta). B. Colocalization (Mander's overlap) between endogenous Arl8b and Rab7 in response to effector depletion, n siC=10, n siRILP=9, n siSKIP=9 images (4≥ cells per image) analysed from 2 independent experiments. C. Immunoblot analysis for depletion efficiency of SKIP and RILP, with actin as loading control.
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B) Fisher exact test to identify significantly enriched GO-terms in the PD-associated proteins in urine. Importantly, the enrichment score of the Fisher exact test does not indicate if the proteins were up- or downregulated in PD patients but rather that the regulated proteins - independent of the directionality - compared to the total urinary proteome are associated with the enriched term. All GO-terms that were significant in both cohorts are displayed (FDR < 5%
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(D) Comparison of the ATP-dependent DNA unwinding activities of WT and mutant TmMR proteins. Lane 2, no protein; lane 3 and 4, WT TmMR; lane 5 and 6, Rad50 (Δ54-56) mutant; lane 7 and 8, R87E mutant; lane 9 and 10, K95E mutant; lane 11 and 12, R94E/K95E mutant; lane 13 and 14, K115E mutant. For each TmMR, reaction contains 1:2 or 1:5 ratio of DNA: protein.
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DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. CFSE proliferation assay (D) among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated with LPS for 48 hours.
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F) Cells generated in (D) were transfected with siCtrl (siC) or siADAR1 (siA). Protein lysates were prepared 78h post-transfection, followed by SDS-PAGE and immunoblotting using the indicated antibodies (n=2).
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A RIP experiments were performed using anti-myc antibodies and extracts from Trt1-myc expressing strains followed by qRT-PCR to detect G-rich TERRA, polyA+ TERRA, ARRET, TER1 and ACT1. Values represent fractions of input RNA detected in immunoprecipitated material expressed as fold increase over untagged (unt) wt strain. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to unt; two-tailed Student's t test).
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A, B, Representative images of scaf1-/- and scaf1+/+ females (A) and males (B) fed with the indicated diets.
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B Intensity of the nucleosome band (nucl) is plotted. The value at 4 µM of yNap1-H2A-H2B (lane 13) was set to 100%. For non-specific DNA binding, the value in the absence of H2A-H2B (lane 5) was set to 100% intensity and disappearance of the free DNA AF647 band was plotted. Each curve is representative of three independent experiments performed on different days. Standard deviations are shown.
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E, Dynamics of MC subpopulations during heart regeneration as shown by the proportion of each subpopulation in total nonCM or total MC.
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A. Superimposition of Rb+/Ba2+ and Ba2+ bound EAAT1CRYST structures with tranD depicted in pink and green, respectively, and TM4 removed for clarity. Black mesh depicts anomalous-difference map in the absence of Rb+ contoured at 4σ around the tranD, and inset shows details of Ba2+ coordination at Na3. Residue numbering corresponds to EAAT1WT, and the coordination distance (angstrom) is parenthesis.
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mRNA expression analysis of epithelial cell differentiation markers Sftpc (AEC II), Hopx (AEC I), Foxj1 (ciliated cells), Muc5ac (secretory cells) and p63 (basal cells) in BALO at days 0, 10, and 21 of culture (n=3 biological replicates with pooled cells from 4 cultures per replicate)
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22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Hemoglobin serum levels were significantly improved in imatinib treated mice.
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A. Scheme to assess the influence of the highly connected nodes on germ layer acquisition.
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HEK293T-ACE2-GFP stable cells were transiently transfected with mouse (m) IFITM plasmids or vector control for 24 h. C) Graph depicts normalized infection percentage measurements from 2-3 independent experiments, each performed in triplicate, from cells infected Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.
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C HSF1WT or HSF1S121A was co-expressed with either GFP or GST-AMPKαCA in HSF1-deficient HEK293T cells. HSF1 Ser121 phosphorylation was examined by immunoblotting.
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(D) TEM images of upper crypt goblet cells from control and Atg5VC transverse colons. The yellow dashed lines outline the mucin granules in the apex of the cells. Bars=2 μm.
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(B) Illustration of the Mic60 redistribution upon re-expression of Mic10 in Mic10 depleted mitochondria.
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Blob plot depicting selected marker genes in cell clusters. Dot size encodes the percentage of cells expressing the gene, and color encodes the average per cell gene expression level.
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B In vitro Ub chain formation assay by Ubc7 with indicated Hrd1 (left) and Doa10 (right) variants in the presence of indicated Cue1 variants. Rates for reactions of mono-Ub to di-Ub and di-Ub to tri-Ub with fluorescently-labeled Ub are shown on a logarithmic scale. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity only significances related to the "no E3" control of given reaction set are shown.
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A HeLa cells were transfected with plasmids encoding CB-GFP or CB-TOP2A-GFP. Lysates of asynchronously growing cells were immunoblotted with the indicated antibodies.
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D Immortalized Brca1F/−MEFs were infected with retroviruses expressing TRF2 shRNAs and/or Cre recombinase, together with a lentivirus expressing PARP1 shRNA, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 was used as a loading control. *non‐specific band.E Quantification of the frequency of end‐to‐end chromosome‐type fusions of cells treated as in (D) represented as a percentage of fusions observed after TRF2 depletion. A minimum of 1,500 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. The P‐value was calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.
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F. RT-qPCR confirmation of transcriptomics results for selected transcripts (MX1, MX2, IFITM1, ISG15 and IFIT1) in A549 cells treated as outlined in (A). GAPDH transcript levels were used for normalization. Data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by unpaired 2-tailed t-test on ΔCt values (*p-value < 0.05; n.s. not significant).
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We included 126 studies in our curated cancer metabolomics dataset, studies that described profiling in multiple cancer types were counted once for each type, giving rise to 136 "cancer vs control" cohorts spanning 18 different cancer types, 71 assessing blood, 39 tissue and 26 urine. We also included 18 diabetes mellitus type 2 studies to determine if our vote-counting method could identify distinct metabolic signatures in cancer versus diabetes.
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(A) The number of AV in neurites decreased significantly in neurons exposed to the SIV-infected microglia supernatant for 3 or 24 hr, however; pretreatment with rapamycin blocked this effect. *** P<0.001, for n = 6 experiments. Scale bar, 20 µm.
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A Permanent LAD occlusion was perfomed at ZT5 or ZT13. TTC staining (white, infarct; red, vital myocardium) and quantification of infarct size normalized to the left ventricle (LV). Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0041.
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B qPCR analysis of CAT genes expression in the third and fourth rosette leaves from 36-day-old plants. The expression of CAT genes in the wild-type plant is given as 1. Three independent experiments were conducted. Data are represented as means ± SD, n=3. Student's t test, *p<0.05.
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Rapamycin treatment recovers survival of TSC1 knockdown in P493-6 cells. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 14 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 30pM rapamycin where indicated (mean ± st.dev., n=3 biological replicates).
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(B) Decreased synaptic PRR7 immunostaining following glutamate stimulation in DIV 21 hippocampal neurons. Epoxomicin (50 nM) added 20 min prior to stimulation did not affect this decrease (Cont=100±3.6%, Glut=32.3±1.3%; +epoxomicin Cont=99.8±4.6, Glut=42.9±1.1). (n=14-17 dendrites, >1200 puncta for each group). Statistical analyses for (A) and (B) were performed using the Mann Whitney test.
|
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B Proportion of protein aggregation following renaturing with the ds/ss/ds oligonucleotides shown in (A).
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(C) Quantitative RT-PCR analysis of selected transcripts in the Sst-Cre::ReteGFP (darker colors) and Ret-eGFPHi:IB4Neg (lighter colors) subsets displayed as differential expression compared to the Ret-eGFPHi:IB4Neg subset. Almost all the genes that are up-regulated in the Ret-eGFPLo:IB4Neg compared to the Ret-eGFPHi:IB4Neg subset are also enriched in the Sst-Cre::ReteGFP population (n=3)
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C Percentage of indicated mutant animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined in each group.
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B Co-immunofluorescence staining of pre- versus post-menopausal tissue for E-cadherin (cyan), PDGFRβ (yellow) and F-actin (pink). DAPI is shown in grey. The arrowheads in (v) depict fibroblasts in direct contact with the myoepithelial layer. For 2D and 3D confocal imaging: n=13 premenopausal and n=10 postmenopausal specimens. Scale bars: wholemount and optical sections:100 μm (panels i-iv); enlargements, 30 μm (panel v).
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E Envelope V1 to V3 region of HIV-1 RNAs in (D) was reverse-transcribed and PCR-amplified. The PCR products were proceeded to TA-cloning. At least 60 single clones were picked from each group and sequenced. These sequences and the standard HIV-1 sequence HXB2 were aligned. Genetic diversity index of each sequence was calculated for each group. F The schematic of CAF-1 body-mediated HIV-1 latency. Detailed information for the schematic was indicated in discussion.
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I. RT-qPCR analyses of GSNOR mRNA in input and heavy polysome fraction pooled together in H2O2-treated samples (for either 8 or 24 h). H3A mRNA expression level was used as housekeeping gene control. Results represent the means ± SD of n ≥ 6 independent experiments shown as % normalized by H3A. ***, p<0.001 Vehicle: PBS.
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D Endogenous, reciprocal co-IP of FOXP3 and TRAF6 in murine iTreg lysate. Naïve CD62Lhigh/CD25-/CD4+ T cells were obtained by FACS and activated with anti-CD3/CD28 antibodies (1ug and 2ug/ml, respectively) in the presence of IL-2 and TGFβ (100U/ml and 5ng/ml, respectively) for four days before lysis. Antibodies against TRAF6 (left) or FOXP3 (right) were used to pull down target proteins and their interaction partners, which were resolved under denaturing conditions and visualized by immunoblotting.
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A and B) The bleach was applied to elongated membrane clusters of the LS‐TVN approximately 27 h post‐infection. The UIS4‐mCherry signal typically did not recover in the bleached area (A), although it was found to recover in some elongated membrane clusters (B).
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(J, K) The H&E staining (J) and Masson's trichrome (K) of the histological sections from implanted NC and CDC20sh hBMSCs scaffold hybrids. Scale bar, 100 μm.
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G) Representative TEM images of longitudinal sections of quadriceps muscle from young and old control and Mfn2KO mice and quantification of mitochondrial area and density (n=3 mice per group) (Scale bar: 0.4 µm). H) Representative TEM images of aberrant mitochondria highly present in Mfn2KO old mice. Data represent mean ± SEM. #p<0.05 Mfn2KO vs. control mice, *p<0.05 old vs. young mice.
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B. BAZ2A associates with chromatin of ESC and NPC. Chromatin-bound (Chrom.) and soluble (Sol.) fractions of equivalent cell number of ESC+2i and NPCs were analyzed by western blot for BAZ2A levels. Total, total protein. Tubulin and histones are shown as loading and fractionation control.
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Wild type mouse cortical neurons depleted of Nprl with shRNA had mitochondria that were insensitive to amino acids (R+L Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein, a downstream target of the mTORC1-S6K pathway. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test
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A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Confocal microscopy of MEFs expressing the indicated GFP fusion proteins or immunolabeled for WIPI2 and infected with mCherry expressing S. Typhimurium (C-H).
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B The flow cytometry figures of each group. The reactivation efficiency was indicated by the GFP-positive cells percentages which were labelled on the top right corner.
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(J) Equal amount (2*105 cells) of doxorubicin-induced (250 nM for 24 hours, right flank) and abemaciclib-induced (4 μM for 8 times 24 hours, left flank) senescent MDF-3MR cells were subcutaneously injected into the same wild-type mice. At 1 dpt, 7 dpt and 15 dpt, the above-mentioned mice were injected with coelenterazine and bioluminescence from the injected MDF-3MR cells was visualized/quantified by the IVIS spectrum in vivo imaging system and quantified (n=4 mice/group). Upper panel, scheme of experimental design. Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.
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0
] |
(I) Lungs were resected and subjected to hematoxylin-eosin (H&E), trichome (collagen) and α-SMA IHC staining (scale bars, 200 μm),
|
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] |
Percentage of GFP+ cells indicative for CRE expression and recombination in the peripheral blood after tamoxifen administration by gavage (5 x 2 mg, 200 μl/mouse; daily). Bars represent means ± S.E.M. (Day 0 N=9/10, WT/Chk1fl/fl, Day 7 N=8/7, Day 14 N=3/3).
|
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] |
C) The efficiency of the removal of the duplicated region at the DNA level was assessed via qPCR analysis by normalizing the signal obtained from the duplication junction to that of the total Dmd signal. Dup18-30 untreated, n = 3-4; Dup18-30 treated, n = 5-9.
|
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] |
(C) Flag‐tagged DAPK (60 ng) was incubated with GST-WT beclin 1 or with GST-T119A beclin 1 (1000 ng) in the presence of Ca2+, calmodulin and ATP for 30 min. GST‐beclin 1 levels were visualized by Ponceau S staining, and phosphorylation on Thr 119 was detected by a phosphoThr 119 antibody (Western blot).
|
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] |
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