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H, I: Binding profiles of OCT4 and NANOG in the proximity of two genes representative of (H) Class I (Tet2) or (I) Class II (Sall1) in EsrrbHi, EsrrbMed or EsrrbNeg E-GFPd1 ESCs. The relative occupancies of NANOG and OCT4 at Tet2 or Sall1 (positions highlighted by hatched boxes) alongside expression levels of Tet2 and Sall1 mRNAs in EsrrbHi (EH), EsrrbMed (EM) or EsrrbNeg (EN) GFPd1 ESCs and EpiSC (Epi) is shown at the bottom. Oct4 binding data from EpiSC
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B. Overlay of three cryoEM maps (grey, blue and green) of the CENP-A nucleosome obtained by sorting on the DNA entry/exit site (Figure EV2F). Distance between most open (grey) and most closed (green) map is 2 Å. The entry/exit site is boxed and the corresponding model is shown on the right. Note that the density of the H2A C-terminus is well defined. C. Overlay of three cryoEM maps (grey, blue and green) of the CENP-A/CENP-CCR complex obtained by sorting on the DNA entry/exit site (Figure EV4F). The distance between the most open (grey) and most closed (green) maps is 9 Å. Map assigned to CENP-CCR is shown in magenta. The entry/exit site is boxed and the corresponding model is shown on the right. Note the absence of a clearly defined density of the H2A C-tail (indicated by the dotted circle).
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A. Characteristics of IGR39 /IGR37 and WM115/WM266.4 melanoma cell lines (Upper Panel). Pigmentation of melanoma cells pellets (lower panel).
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A. Lysates of HEK293T cells, transfected with the indicated plasmids, were subjected to immunoprecipitation (IP) with the FLAG antibody (PSMA2), followed by immunoblotting (IB) with the myc antibody (FBXO7).
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(C) Body weight; (D) Heart weight and (E) Heart-to-body weight ratio (n = 28-40).
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PolyUb proteins (FK1) labelling in HeLa cells left untreated or treated with HS at 42˚C for 2 h, alone or combined with CHX (50 μg/ml). Scale bars: 5 µm.
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B-D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 (EP1i, SC 51322), EP2 (EP2i, PF 04418948), EP3 (EP3i, L-798,106) or EP4 (EP4i, L-161,982) at a concentration of 10 µM or an equivalent volume of DMSO vehicle. **p<0.01, ***p<0.001, ****p<0.0001 compared to the DMSO only group by one-way ANOVA and Dunnett's post-test. (D) Quantification of the average expression ± s.e.m. of Cldn4 mRNA relative to stem cellspheroids (n = 3 independent experiments).
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(C) Detection of decoy antibody ACE2-Fc and Spike S1-Fc chimeric homodimer formation in nonreducing Coomassie Brilliant Blue staining.
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C-F. Representative, exposure matched maximum projection confocal images of FISH for Kpnb1 (C) or mTOR (E) mRNA and NF plus Tuj1 immunostaining from sciatic nerve sections from WT (left) or GAR+/- mice. Upper panels for each show total mRNA signal. Middle panels show mRNA (gray) signals merged with NF plus Tuj1 (magenta) and DAPI (blue). Lower panels show mRNA signal that overlap with NF plus Tuj1 signal (labeled "axon only" signal). Quantification of axonal Kpnb1 (D) and mTOR (F) mRNA signals compared to the negative control, DapB mRNA, show a significant reduction in these axonal mRNAs in the GAR+/- mice. n = 3 WT, n = 3 GAR+/-; means ± SEM; ** p < 0.01, *** p < 0.001, ****p < 0.0001 unpaired Student's t-test. Scale bar - 10 µm.
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(G) Boxplot showing resistance to 3mM H2O2 grouped by pyk1 allele for 156 strains from our collection. The T-strains had a higher mean fitness in H2O2 than the A-strains (1.01±0.06 vs 0.95±0.11; p=0.0021, Welch's t-test). The resistance score was obtained as for antimycin A.
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(E) A heatmap showing relative mRNA expression levels of 2,137 genes differentially expressed (>2-fold) between Yap1 OE ES cells and control ES cells. Genes were sorted by the fold changes of gene expression between Yap1 OE ES cells and control ES cells (first column) and corresponding gene expression profiles obtained from dESC are shown.
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FLIP experiment showing a bleached (FRAP, red circle) and unbleached, adjacent locus (FLIP, blue circle) in the same nucleus. The graph displays average relative fluorescence of FLIP (blue), FRAP (red), and control loci (no bleach, green) in other non‐bleached cells.
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B RNA pull-down assay using biotinylated major and minor satellite RNA transcripts and whole cell extracts from Miwi heterozygoous (Miwi+/-), knockout (Miwi-/-), slicer activity-deficient mutant (Miwi-/ADH), and Mov10l1 germline knockout (Mov10l1f/-;Stra8-Cre) testes. Mov10l1f/-;Stra8-Cre mice represent a pachytene piRNA-deficient condition. Approximately one repeat of In vitro transcribed major and minor satellite RNAs corresponding to the sequences in A were used as baits. Precipitated proteins were visualized by Western blotting using antibodies against the indicated proteins (MIWI and MILI). The input equals 1/10 to 1/80 of the protein lysate used in the pull-down. Transcripts corresponding to 300 nt and 162 nt of the 18S rRNA sequence served as controls for the antisense (As) and sense (S) sequence of major and minor satellite transcripts, respectively. The blot was re-probed with anti-GAPDH antibody serving as a negative control.
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A Schematic diagram of MAPKKK5 constructs. N; N-terminal domain, KD; kinase domain, C; C-terminal domain.
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G Protein extracts of scramble or USP19-specific siRNA treated 293T cells transfected with HA-MAVS plasmid along with vector encoding for Flag-RIG-I (N) in the absence or presence of Beclin-1 were subjected to immunoprecipitation and immunoblot analysis.
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e, Co-treatment with chloroquine (CQ, 10 µM) showed that the decrease in LC3 ratio was a result of an increase in autophagic flux.
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E. Schematic diagram showing D-Asp exposure protocol in organotypic slices after LPC exposure
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(A) DIV 3 cortical neurons immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (B) DIV 8 cortical neuron transfected with GFP and immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (C) DIV 16 cortical neuron transfected GFP and immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (D) Somatic PIP3 quantification in the soma at increasing days in vitro. Data are shown as the mean +/- SEM. P values are significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 neurons. (E) Growth cone PIP3 quantification at increasing days in vitro. Data are shown as the mean +/- SEM. P values indicate significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 growth cones.
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B) Bar graph representing RNA levels as in Fig. 1D, relative to a control locus (act1) and to WT levels, as measured by qPCR. Error bars represent standard deviation of nucleosome occupancy of biological duplicates.
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(H) Both liver-specific (lfabp promoter; n=46) and widespread (bactin promoter; n=18) overexpression of igfbp1a increase β-cell regeneration when compared to control (n=77).
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e, Correlations between individual cell cycle phase durations. Sample sizes were adequate to detect correlations n = 125 (RPE), 130 (U2OS), 113 (H9). R2, square of Pearson correlation coefficient.
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A ChIP analysis was performed on wild type (untagged) and abo1-GFP cells and the resulting DNA analysed by qPCR for centromeric (dh, dg and imr) repeat sequences. Data are the mean of four independent biological repeats and error bars represent ±SEM. P-values calculated using a two-tailed unpaired t-test indicates that all loci are significantly enriched (P < 0.05) relative the untagged control.
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(A) Schematic representation of the study design and Western blot analysis for TIP30 and GAPDH in hearts from TIP30 wild-type (WT), heterozygous (Het) and homozygous knock-out (KO) mice under basal conditions.
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(F) Representative western blot of scr or miR-29b transfected TREx cells analysed for ORF65 expression with GAPDH as a loading control. Densitometry analysis performed on n=3. Data information: data are presented as mean ± SD. **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.
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(B) Meta-gene profile of p65 ChIP-seq signals (rpm/bp) from the TSS to TES and +/- 5kb on BRD4 independent genes (top) and BRD4 dependent genes (bottom).
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(E) Mitochondrial oxygen consumption rate (OCR) measured in Ctrl and ORP5 siRNA transfected cells. OCR trace was obtained by sequential measurement of basal OCR (OCRBAS), OCR after the addition of oligomycin (OM) and OCR after addition of antimycin A (AA). Note the reduced basal OCR (OCRBAS) compared to ctrl siRNA cells. Error bars denote ±SEM. Data are the mean of 4 independent repeats (n=4), *P<0,05.
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A-C Quantification of bacterial DNA in the gonadal white adipose tissue (A) (WAT,n = 14-16 mice for all groups), mesenteric adipose tissue (B) (MAT,n = 10-15 mice for all groups), and liver (C) (n = 10-12 mice for all groups) of chow-fed and 16 week HFD-fed WT and NOD2−/− mice, *P = 0.0002.
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Lys-1034 is a major target of DSB-induced sumoylation of separase. Hek293T cells expressin Myc-separase-WT or -K1034R (D) were DRB- or mock-treated and subjected t Myc-I Input samples and eluates were immunoblotted using the indicated antibodies
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The increased total and Ace-β-cat (K49) levels positively correlated with increased phosphorylated Tau pS199 and AT8 as detected by Western blotting in the hippocampal extracts of AD patients compared with age-matched controls (n=10 each group Pearson analysis was used for correlation analysis
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WT mice were acclimated to cold for 21 days (21d, 4ºC) and then deacclimated at thermoneutrality (29ºC) for 1 day (1d) or 7 days (7d). Representative immunoblot of Parkin phosphoSer65 and total Parkin in whole lysates of iBAT and after Parkin immunoprecipitation. Arrowheads indicate Parkin 52kDa band and asterisk indicates a non-specific band.
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c. Correlation of the average expression of 163 genes across major cell types between MERFISH measurements to scRNAseq data from NCTT. d. Correlation of the average expression of the same genes as in c between expression of types in scRNAseq data from NCTT. The correlation structure in panel C closely mirrors the structure in panel D.
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H Detection of splenic TNP-KLH specific IgM (left) and IgG (right) -secreting cells by ELISPOT assay (n=7 NC, 4 CPA300, 3 WT, 2 CD40LG). Spots were counted by an ELISPOT Reader using a size range of 0.005-1 mm. Median ± IQR.
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(A) Analysis of the cell growth rate of control and subject fibroblasts, overexpressing or not an EGFP-tagged version of the wild-type SLC25A46. Experiments were done in independent triplicates. Error bars represent mean +/- SEM and p-values were calculated using t-test.
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(G-H) Quantification of normalized MA currents recorded from large DRG neurons dialyzed with 50 ng/ml Gβγ (G, n=7) or with 0.0001% Lubrol (vehicle for Gβγ) (H, n=6). *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.
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D. Fluorescence microscopy, green fluorescence absorbance, and virus replication at 24 hpi in RIG-I-/- MEFs. Cells were transiently transfected with empty vector, RIG-I wild-type, RIG-I K909Q, or RIG-I K909R mutant for 24 h, followed infection with VSV-GFP (MOI = 1). bar, 100µm
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B. Basolateral release of 14C-labeled fatty acids in Caco2 cells expressing shscramble or shPkd2 in a transwell system and treated with CRT0066101 for 24 hours. n=8.
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Nlrp3-/- BMDMs transduced with lentiviruses expressing Flag-NLRP3 (WT) or Flag-NLRP3 (A350V) were left unstimulated or stimulated with LPS or LPS plus ATP. Immunoblot analysis of NLRP3 and NLRP3 (A350V) ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.
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EMT-related markers, stemness markers and drug resistance genes were evaluated by real-time PCR (C) in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control.
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A. Cartoon representation of the IFT22-binding site on IFT74/81 ccVI (top left) and surface conservation representation of different orientations of IFT22/74/81 (top right and bottom). IFT74/81 ccVI displays a highly conserved patch at the IFT22-binding interface (black dashed circle). A 180° rotation of IFT22 (bottom right) exhibits a likewise conserved patch at the IFT74/81-binding interface (black dashed circles; position of the IFT74/81 helices is marked with light grey lines). Conserved residues are marked and labeled according to the Tb sequence. Conservation coloring is based on Clustal Omega multiple sequence alignments with H. sapiens, M. musculus, D. rerio, T. brucei, C. reinhardtii and C. elegans sequences and ConSurf conservation grades
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B-C. (B) Micronuclei frequency formation by live cell imaging in untreated (NT) and Auxin-treated (IAA) condition. (C) Bar graph represents the type of chromosome mis-segregation (independently of the dCas9 signal) observed in the indicated conditions, with (IAA) or without (NT) auxin, in the cell lines expressing dCas9 mScarlet-I and sgRNA targeting chromosome 1 or 3. Error bars represent the SEM of four independent experiments in which cells labeled for chromosome 1 and 3 were analyzed together (n = 45 - 149 cells).
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(A) CSAG2 does not alter SIRT1 protein levels. HEK293FT cells were transfected with different amounts of Myc-CSAG2 for 48 hr before cells lysates were harvested and immunoblotted for indicated proteins.
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Transcriptional activities (Tx) of 421 regulatory sequences that were active in all 10 bacterial cell-free expression systems.
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D Western blot of the GSTpull‐down using antibodies against Matrin3 and Raver1. Lanes 1 and 2 show 5 and 10% of input, respectively, lanes 3 and 4 show GST‐PTB full‐length pull‐down of wild‐type and Y247Q mutant, respectively, and lanes 5 and 6 show GST‐PTB RRM2 pull‐down of wild‐type and Y247Q mutant, respectively, and lane 7 with pull‐down using GST alone.
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b. H&E-stained skin sections of wound-induced papillomas of InvEE PRSS35+/+ and InvEE PRSS35-/- mice. Scale bars: 100 µm.
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(a) Histogram showing relative phospholipid levels in CHO cells treated with 0.3% ethanol for 30 min before cell harvesting. Total PLD activity is measured as phosphatidylethanol (PEtOH) levels under the following conditions: normal medium (Nm), serum-free medium (SFM), serum-free medium with 100 nM wortmannin (SFM+W), nutrient deprivation (0.5 h St; 1.5 h St; 3.0 h St), 30 min of nutrient deprivation in presence of 100nM wortmannin (0.5h St+W). Other phospholipids were used as controls: phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC). Values denote means±s.e.m. (n=9, except for SFM+W and 0.5 h St+W, where n=3 and 5, respectively). **P0.01; *, P0.05.
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(D) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed mouse hepatocytes were treated or not with latrunculin (LatA) as indicated, extensively washed to remove traces of the inhibitor, and then labelled with the antibody and subjected to in vitro fusion assay in the presence or absence of purified actin. The number of total fusion events/total number of vesicles for each condition was as follows: 176/880; 233/1110; 84/930; and 180/950. The differences with untreated samples were significant at **P0.01.
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H Schematic illustration of the role and translational scope of the miR-34a/Pdgfra interaction during arrested lung alveolarization. Hyperoxia drives miR-34a expression in myofibroblasts, downregulating PDGFRα expression and reducing PDGFRα+ cell abundance, causing the perturbed elastin fiber production and blunted alveolarization seen in bronchopulmonary dysplasia (BPD). The effects of hyperoxia are attenuated when miR-34a function is blocked with an antimiR (A34a) or when the miR‑34a/Pdgfra interaction is disturbed with a target site blocker (TSB).
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a. Log2 fold change of probe intensities of PRSS35 gene expression in different subsets of dermal fibroblasts observed in Affymetrix GeneChip® Mouse 430 2.0 array (n=3 per condition). b. Hexagonal triwise plot depicting the PRSS35 gene expression level.
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O, P Double immunolabeling of PDS5A (green) and kinetochores revealed with an ACA serum (red) in a metaphase I spermatocyte. Data information: DAPI staining of the chromatin (blue) is shown for some spermatocytes.
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G. CIV activity as measured using an oxygraph Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g
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Top: Schematic of the co-culture system used to assess vesicle transfer from donor (labeled with DiD) to acceptor (labeled with CTG) cells. After labeling, cells were detached, replated in 1:1 ratio and cultured in control conditions or treated with 200 µM H2O2 or 200 ng/mL Wnt7a for 4 h. Middle: Representative confocal images of co-cultured donor and acceptor CAD cells. Each image corresponds to the projection of the entire Z-stack. Bottom: The insets correspond to the projection of selected Z-stacks of the area marked with a yellow square in each condition to better illustrate the transferred vesicles in acceptor cells. Yellow arrows point to DiD-labeled vesicles inside acceptor cells. Percentage of acceptor cells containing DiD-labeled vesicles in the co-culture systems with the indicated treatments.
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A, B. (A) IgG and (B) IgM responses were analysed by screening plasma samples of asymptomatic (n=39, median study day 29) and symptomatic (n=57, median study day 31) COVID-19 patients. Data are presented as Mean ± SD ***P<0.001 (Mann-Whitney U test).
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Reverse co-IP confirmed the interaction between SAMHD1 and TRIM21. SAMHD1-flag was transfected with TRIM21-HA into HEK293T cells, and then the cells were treated as in (B) and subjected to HA IP and IB.
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(A-C) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 mins. WT n = 330 cells, N = 4 embryos; Cep63T/T n = 260 cells, N = 3 embryos; Sas4cKO n = 223 cells, N = 3 embryos. (D-F) Percentage of proliferating, arrested and apoptotic progeny within each group of the indicated mitotic duration, calculated from data shown in A-C; groups with n < 20 cells were excluded from this quantification; #, chi-square test with *, post-hoc analysis, within each genotype, comparisons are made to the 0-30min group.
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C, D. Representative confocal images of mitochondrial fusion in Drp1-/- 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of exogenous hFis1. Drp1-/- cells co-transfected with mito-PAGFP, mito-DsRed, and either empty vector (the upper panel) or hFis1 (the lower panel) were photoactivated in a small region of interest (ROI) (white circle, 3 µm diameter) as indicated in preactivation images of mitochondria seen by mito-DsRed (red). After photoactivation, Z-stack images with photoactivated GFP (green) and mitochondrial marker (mito-DsRed) were collected at times 40 sec, 15, 30, 45 min as indicated (C). Mitochondrial fusion was quantified by analysis of changes in the fluorescence intensity of photoactivated mito-PAGFP in ROIs at 40 sec, 15, 30, 45 min after photoactivation. The dilution rates (percentage) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation (D).
|
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g-j, Histological analysis of Atg7flox/+; nestin-Cre (left) and Atg7flox/flox; nestin-Cre (right) cerebellum at P56. Cryosections were stained with HE (g, h) or immunostained for the Purkinje marker calbindin (i, j). Arrows in g and h indicate cerebellar Purkinje cells.
|
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(D) OCR was measured in cells grown in substrate-limited medium and treated in the absence or presence of Etomoxir (Eto). Oligomycin (OA), FCCP (FC) and a mixture of rotenone and antimycin A (RA) was added as indicated. Data are shown as mean ± SEM (n= 69 technical replicates)
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H Evaluation of murine peritoneal permeability six hours after treatment with Control-Fc or Spike RBD-Fc, or seven days after treated with Control-Fc or Spike RBD-Fc daily by TRITC-dextran dye extravasation assay. For each group, n = 8. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. For (H), P values are determined by Student's t-test
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Systematic summary of brain-enhanced expressed proteins and signaling cascades significantly altered in COVID-19 patients (neurotransmitters transport, synthesis). Values for each protein at all analyzed samples (columns) are color-coded based on the expression level, low (green) and high (red) z-scored FOT.
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F-G) H8 or H3 treated cells were analyzed under super-resolution microscopy shows actin protrusions with Tie2 clusters at the tip. Lower panels are Imaris reconstructed 3D model of the super-resolution images. Scale bars are 5 µm.
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(B) Rab8A and Galectin-3 positive vesicle numbers were analysed by immunofluorescence. Data show the mean ± SEM of biological replicates. Scale bar = 20 μm. ns=non-significant, **p≤0.01, by One-way ANOVA followed by Sidak's multiple comparisons test.
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Representative confocal micrographs show EphA2 (red), RSK (green: top) and GPRC5A (green: bottom) in frozen sections of HGSC patient tumors. S indicates the stroma. Scale bars: 50 μm.
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Collision-induced dissociation of the parent molecular ions for m/z 1046.477-3 from Glu-tRNAGA produced expected -c, -w and -y ions for the sequences CU[mcm5s2U]UCACCC
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D. Lifeact-GFP-expressing cells were treated with the indicated PMOs and imaged every minute over 1 hr. Sequential image frames, from a control cell, highlight edge retraction (red arrowheads) or protrusion (yellow arrowheads). Corresponding edge velocity is shown, with negative values indicating retraction and positive values indicating extension. Average protrusion and retraction speeds were calculated from n=11-13 cells. Bars: mean ± s.e.m..See also movies EV2 and EV3.
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(A) NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 30 and 0.01, respectively. At 48h.p.i., the cells were fixed and permeabilized prior to immunostaining.
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C-E Distribution of gold-labeled ATZ-HA by IEM in WT MEF. ES, ER subdomains. Red asterisk shows site of EV:EL membranes fusion. Scale bar: 1 μm.
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G Scatter plot showing the genes which are commonly altered upon R5020 stimulation (adj. P-value < 0.05) in both mouse and human organoids relative to their CTRL samples. Color scale as a function of the averaged human and mouse logFC values. The red dashed lines represent an adj. P-value threshold of 0.001.
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A) Top. Low magnification merged confocal images of coronal striatal sections showing ChAT-labelled (cyano) ChIs in the area of mCherry fluorescence (red) indicating viral infection. Bar = 100 µm. Bottom. Higher magnification images of the ChIs identified by the white box in the upper images show viral-induced expression of mCherry and GFP signal. Bar = 20 µm. B) The mCherry/ChAT overlap is increased in ChIs from Tor1a+/- mice (Tor1a+/+ n=24 cells, Tor1a+/- n=22 cells, N=8 mice/genotype, t test ***P=0.0005), but the mCherry/GFP fluorescence ratio indicated that the LC3 flux was unchanged (Tor1a+/+ n=19 cells, Tor1a+/- n=21 cells, N=8 mice/genotype, Mann Whitney test P=0.2334)
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B. Quantification of YFP-S. aureus average fluorescence intensity per cell from Figure 5 panel (A) (n = 66-85 cells). Data information: One representative experiment out of three shown. Error bars represent SEM. ** P < 0.01, *** P < 0.001, **** P < 0.0001, not significant (ns). one-way ANOVA.
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B. PAS staining (purple) of the nasal mucosa of E18.5 embryos of the indicated genotype. Normal PAS positive goblet cells (inset, red arrows) are interspersed between MCCs (inset, black arrows) with their characteristic goblet or cup-like shape in Gemc1+/+ embryos. The Gemc1-/- mucosa has abundant PAS positive materials that accumulate in the apical borders of almost every columnar epithelial cell (inset, red arrows) and lacks typical goblet cells. Scale bars = 20 µm.
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E: cylinder test. The experiment was carried out on the same group of animals as in panel b. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. *** p<0.001.
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(L) Western blots for Ace-H4-K5, Ace-H3-K18 and H3 in WT and MZsinhcaf -/- FG follicles. α-tubulin was used as a loading control. Data information: FG, full-grown stage (late stage III)
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B. Schematic of RNF43 constructs in which the extracellular and transmembrane domains (ECD and TM) are replaced by those of the unrelated CD16 and CD7, respectively. CCR; CD16-CD7-RNF43.
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C. Ectopic expression of AGO2 restores P body formation, but fails to rescue the impairment in miRNA function in Nup358 depleted cells. HeLa cells were transfected with control (siContriol) or Nup358 (siNup358) specific siRNA, followed by indicated constructs. Right panel: Western blot analysis for the relative expression of indicated constructs in HEK293T cells. a-tubulin is used as loading control.
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c. Representative photomicrographs of larval neuronal cell bodies expressing the matrix-QC reporter and RNAi for mdy or control driven with nSyb-GAL4. GFP is shown in green, mCherry is shown in magenta. Mitolysosomes (mCherry-only puncta) are shown in greyscale. Scale bar = 5 μm.
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G) (Top) Western blot analysis of CrizR1 cells treated with siScrambled or with a pool of 4 different siRNAs targeting Survivin (siBIRC5). (Bottom) Cells were stained with Annexin V/PI and analysed by flow cytometry for Annexin V+ cells 72h post-transfection. Annexin: early apoptosis P =0.0003, late apoptosis P =0.5, alive P <0.0001.
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C Mice (n=3) were intravenously injected with the high salt solution (3% NaCl) or control salt solution (0.9% NaCl, Ctrl). Then mice were intraperitoneally infected with VSV (1x109 PFU per gram body, 12 or 24 hrs) either 12 hrs post injection (P.I.) or immediately (0 hr P.I.). Virus titers in the blood were analyzed by the TCID50 assay. Data information: Data represent mean and SEM of three biological replicates For all statistical testing, p values were calculated using two-tailed unpaired Student's t-test. NS, not significant (p > 0.05). *p < 0.05, **p < 0.01 and ***p < 0.001.
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H, I. Mean 2f1-f2 DPOAE thresholds (± SD) are plotted for wt (green), heterozygote (blue) and homozygous (red) mice aged 4 weeks (H) or 21 weeks (I), as a function of f2 frequency.
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B. Representative RFP immunostaining from the liver of Tie2-GFP mice (left panels) or Tie2-IFNα mice (right panels) at the indicated time points after 5x105 CT26-RFP intrasplenic injection. Arrowheads highlight single or clustered RFP positive cells; scale bars=100μm.C. Immunostaining quantification of hepatic CT26-RFP arrival (5 minutes post-injection: Tie2-GFP n=6, Tie2-IFNα n=2) and expansion (day 3 post-injection: Tie2-GFP n=6, Tie2-IFNα n=3; day 7 post-injection: Tie2-GFP n=5, Tie2-IFNα n=3) in the liver of Tie2-GFP mice or Tie2-IFNα mice that were injected with 5x105 CT26-RFP as described in B. Data pooled from two independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test. The increased percent of RFP+ areas in the liver of Tie2-GFP mice was statistically significant (p=0.0009 by one-way Anova test, not reported on graph).
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C-G) Representative images showing that Hh staining normally localised to a ring like structure (yellow arrows), becomes delocalises to the glial cytoplasm upon htlACT overexpression, quantified in (E) (n=4, 6 brain lobes). Glial cells are marked with repo-GAL4>GFP. (E-F') are zoomed in images of (C-D').
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Immunoblot analysis of WT, NLRP3 KO (F) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.
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quantification of germband macrophages in control embryos and those expressing different RNAis against mitochondrial OxPhos Complex III (or an RNAi against Complex V. For (H) control (n=34 embryos) vs Complex III RNAi 1 (n=20) p=0.0001; vs. Complex III RNAi 2 (n=18) p=0.027; vs. Complex III RNAi 3 (n=16) p<0.0001; vs. Complex V RNAi (n=14) p<0.0001. Data information: show Stage 12 embryos. Complex III RNAis target Cyt-c1, UQCR-cp1-2, for Complex V, ATP synthase F1F0. Mean±SEM, **p<0.01, ***p<0.001, ****p<0.0001. one-way ANOVA with Tukey's (H).
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F. ZNF853 localized to the nucleoli in U-2 OS cells (HPA067690). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.
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Lipid overload in autophagy-insufficient state (Atg7+/−) leads to increased accumulation of lipid due to defective lipophagy. Autophagy insufficiency causes delayed turnover of mitochondria and mitochondrial dysfunction, which leads to augmented inflammasome activation when challenged with inflammasome activators such as lipid. Combined effects of increased lipid content, augmented inflammasome activation and their interaction lead to deteriorated insulin resistance and diabetes.
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SPPL2c protein levels were analysed in germ cell populations from testis of wild type mice sorted for their DNA content which was determined by Hoechst 33342 staining as depicted in Fig EV4D. While for 4C, 2C and 1C fractions 10 µg of protein were loaded, for the testis control samples (=total lysates) 20 µg of protein were subjected to electrophoresis. *, non-specific band.
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(C) Association between IKBKE (IKKε) and PSAT1 mRNA overexpression evaluated by a chi-squared independence test. The + sign indicates samples with significant (p<0.05) overexpression of IKBKE or PSAT1. Number and percentage of samples, as well as the Chi-square values are shown.
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(F) IHC analysis of the islets from Leprdb/db and Lepob/ob mice at the indicated ages using anti-Pdia4 antibody and hematoxylin. Scale bar = 100 μm. The dash circles indicate islet regions. Data information: Data from 3 experiments are presented as the mean ± SD.
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(G) Representative confocal images of β-catenin and UBTD1 in DU145 under resting (Ctrl), after calcium chelator treatment (EGTA) and recovery (Rec) conditions. Nuclei were stained with DAPI (Blue) on the MERGE image.
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Western blot analysis showing depletion of ORP3 in the ORP3−/− hTERT-RPE1 cell clones. GAPDH served as a loading control.
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6-week-old male CD36 knockout mice were administered with AAV-TBG-wildtype-CD36, wildtype-CD36 plus KLF10, palmitoylation site mutant CD36, mutant-CD36 plus KLF10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (E) Immunofluorescence staining of CD36 and β-catenin. (F) Quantitation of CD36 positive area. (G) Co-localization rate of CD36 and β-catenin. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA
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A,B RPE cells expressing shRNAs specific to lamin A/C or nesprin 2 (shNesprin 2, clones #1 and #2) were stained for G-actin with DNase I (green). Representative images are shown (A). Scale bars, 20 μm. The proportion of nuclear G-actin fluorescence intensity to the whole cell was measured (B, n ≥ 67).
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single-cell quantification related to the data in a and b. (c) cytoplasmic [Ca2+] after ionomycin; (d) maximal cytoplasmic [Ca2+] after TG; (e) increase rate of cytoplasmic [Ca2+] when extracellular Ca2+ is applied; (f) decrease rate of cytoplasmic [Ca2+] when extracellular Ca2+ is removed in NKT1 and NKT2 cells which have similar Ca2+ plateau level (iso-cells; 900-1200nM for control group (a), and 400-700nM for CCCP group (b)).
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(d) HeLa cells stably expressing GFP-VPS35 WT and D620N were depleted of endogenous VPS35 using 40 nM of siRNA, and subsequently immunostained for TGN46 and endogenous ATG9A and subjected to confocal microscopy. (e) Colocalization between TGN and ATG9A is expressed in terms of the Pearson's Coefficient. n=25 cells (WT) and 33 cells (D620N). Error bars represent s.e.m. and **P=0.01 by 2-tailed Student's t-test. Scale bars in (a-d), 10 μm.
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G. Representative western blot showing changes of overall O-GlcNAcylation (upper panel), phosphorylation status of each target protein (middle panels) or pro-caspase-3 cleavage (lower panel) in THP-1 cells treated with Stx2a (10 ng/mL) for 9 h in the presence or absence of overexpression of Akt (wild-type or T305A/T312A). Data information: Error bars for bar graphs are presented as mean ± S.E.M. 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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C The distribution of endogenous TFG was examined in cells stably expressing low levels of mRuby‐Sec23A (Sec23A) using SR‐SIM (n = 10). Scale bar, 5 μm. Inset scale bar, 1 μm.
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A-B. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (A) wild type; (B) ADAM17-/-, both reared in the dark. Scale bars: 10μm.
|
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(i) Immunostaining of 5caC shows that TCF20 depletion increased 5caC level. White dashed lines show the cells infected with lentivirus. Scalebar 25 μm. (j) Statistical analysis of the 5caC fluorescence level. n=20 cells, and P<0.0001. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***).
|
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A. Linear representation of the structure of EML4-ALK V1 and V3 variants and the deletion 12N blade mutant including residue numbering indicating domain boundaries.
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I-J. Quantification of capillary number per area (mm2; I) and average of capillary cross-sectional area (µm2; J) in mdx-Control and mdx-CriptoMy-LOF diaphragm muscles. Data are mean±SEM (n=5 biological replicates; ***P<0.001, Student's t-test).
|
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A, B and C. Changes in mRNA expression of Ucp1 (A), Pgc1α (B) and Pparγ(C) from WT and TRPV2KO iBAT 4 h after intraperitoneal administration (i.p., arrows) of saline or a selective β3-adrenergic receptor agonist, BRL37344 (600 µg/kg body weight). Mean ± SEM, n = 5 - 7; * P < 0.05 vs. saline group; # P < 0.01 vs. WT BRL37344 administration group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.
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