EMBO/sd-character-level-ner huggingface dataset:
HXT mRNAs are retained in the mother before MAT, but only HXT2 mRNA is enriched in the bud after MAT upon refeeding.Figure 4A Table of the glucose affinity of the four most important hexose transporters. B FISH of HXT1, 3 and 4 under glucose rich conditions. White arrowheads depict cells before, yellow arrowheads cells after MAT. C Quantification of (B). D Hyperactive PKA promotes enrichment of all HXTs after MAT. E HXT2 mRNA is the only HXT mRNA showing enrichment in the bud after MAT upon transient PKA activation. F Elutriation confirms enrichment of HXT2 mRNA in daughter cells upon refeeding. Daughters and mothers were separated through elutriation. qPCR was performed to determine the relative levels of different HXTs and ASH1 mRNA in both fractions. G Enrichment of HXT2 mRNA in the bud leads to higher Hxt2-GFP transporter levels at the plasma membrane of the daughter cells. Daughter cells were enriched through elutriation. Quantification of the fluorescence intensity of the plasma membrane from life cell imaging from three independent experiments in which at least 50 cells were analyzed per condition and experiment. Western blot analysis confirms the higher amount of Hxt2-GFP transporter protein present in daughter cells under glucose-shift conditions
Figure 3A Schematic representation of signaling pathways targeted by the inhibitors used in this study. B eEF2K WT and KO MEF treated for 6h with indicated concentration of NFR, Rapamycin (Rapa), AICAR or starved for indicated time in PBS (Starv.), were analyzed by immunoblot using indicated antibodies. Tubulin is used as loading control. C A potent mTOR inhibition does not impair NFR-mediated eEF2 phosphorylation. WT MEFs were treated with the indicated concentrations of Rapamycin or with vehicle (Mock). After 30 minutes, indicated doses of NFR were added and cells were incubated for additional 6h and analyzed for phosphorylated S6R and eEF2. Tubulin is used as loading control. D NFR-mediated eEF2 phosphorylation is not affected in AMPKα1α2 dKO. AMPKα1α2 WT and dKO MEF treated for 6h with indicated concentration of NFR, Rapamycin (Rapa), AICAR or starved for 1h in PBS (Starv.), were analyzed by immuoblot with indicated antibodies. * anti-total AMPKα antibody give an unspecific band with a slightly higher molecular weight in dKO cells. Tubulin is used as loading control. Each panel is representative of at least 3 independent experiments.
Figure 3A. Schematic of WDTC1 knockdown-rescue lentiviral vector (top). 3T3-L1preadipocytes were lentivirally transduced to generate stable cells expressing a non-specific (NS) shRNA control, shWDTC1 targeting endogenous Wdtc1mRNA, or cells simultaneously expressing shWDTC1 and shRNA-resistant WDTC1; bottom, confirmation of knockdown and WDTC1 ectopic expression. B-D. 3T3-L1 stable cells described in (A) were adipogenically induced. Their adipogenic potential was assessed by ORO (B) B-D. 3T3-L1 stable cells described in (A) were adipogenically induced. Their adipogenic potential was assessed by triglyceride quantitation (C) B-D. 3T3-L1 stable cells described in (A) were adipogenically induced. Their adipogenic potential was assessed by adipogenic gene expression (D).
Figure 4(A) Streptozotocin-induced diabetic DEREG C57BL/6 mice were treated with mouse recombinant IL-33 daily for 5 consecutive days, as well as diphtheria toxin (DT), PC61 or DT+PC61 on days -4 and -1 prior to and on day 2 post islet transplantation. Mice were sacrificed at day 80 post-islet transplantation or at the day when grafts were considered rejected. Proportion of CD4+Foxp3+ Tregs (B) from the spleens and kidneys of islet transplant mice receiving vehicle, IL-33, IL-33/DT, IL-33/PC61, or IL-33/DT/PC61 at day 5 post-islet transplantation. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed; NS: non significant, ***P<0.001. Proportion of Lin-GATA-3+ ILC2s (C) from the spleens and kidneys of islet transplant mice receiving vehicle, IL-33, IL-33/DT, IL-33/PC61, or IL-33/DT/PC61 at day 5 post-islet transplantation. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed; NS: non significant, ***P<0.001. (D) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. *P<0.05, **P<0.01. (E) Data are shown as blood glucose measurement in islet transplant mice treated with IL-33/DT, IL-33/PC61, or IL-33/DT/PC61. The horizontal black line indicates a BGL of 16 mmol/liter, the threshold for rejection. Each line represents one mouse.
Figure 3(A) Correlation function of H3K4me3 ChIP-seq reads. A broadening of H3K4me3 domains in CLL by 1-2 nucleosomes was detected. Error bars represent the SEM. (B) H3K4me3 peak width distribution at common promoters in CLL and NBC controls. (C) Distribution of nucleosome occupancy calculated from histone H3 ChIP-seq averaged over a 1000 bp window within promoters. H3K4me3 regions displayed higher nucleosome density for CLL as compared to NBC samples. (D) Exemplary region at the TAF13 promoter showing higher H3K4me3 levels upstream of the TSS with lost ATAC signal (grey bar) as compared to the NBC control. (E) TF motif analysis of ATAC signal lost at CLL promoters with broadened H3K4me3 regions. An enrichment for the NFY TF motif was detected. (F) Heatmap of genome-wide histone modification patterns at promoters within -2 to 5 kb around the TSS (x-axis) for an NBC (H3) and a patient (CLL1) sample. Each line on the y-axis corresponds to one promoter. The clustering revealed one cluster with loss of H3K4me3 at bivalent promoters in CLL. (G) TF motif analysis at bivalent promoters that lost H3K4me3 in CLL.
Membrane tethering by the Atg5–Atg12/Atg16 complex.Figure 5(A) Frames of widefield images taken from a time series of rhodamine‐PE labelled GUVs in the presence of the Atg5-Atg12/Atg16 complex. For the control condition buffer but no protein was added. Note the increase in clustered GUVs over time. The numbers indicate the time in minutes. (B) Confocal images of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP complex. Note the accumulation of the complex at the interface between GUVs (arrows). (C) Individual confocal microscopy frames of a time series of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP. The arrows point to the GUV-GUV interface where the protein initially accumulated. (D) Confocal microscopy images of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP. The arrows point to the concentrations of Atg5-Atg12/Atg16 on the GUV membrane. Scale bars: 10 μm (A-C) and 3 μm (D).
Fig.Figure 3(A) Na+ density from MD simulation (m2-00-f-3), measured in mol/l. The bulk density is ~150 mM. Only one binding site is visible with this cut through the density. The membrane is omitted for clarity. (B) Top-view of the binding site; Na+ positions are shown as small spheres, drawn at 10-ns intervals. Yellow/cyan/solid gray: simulation m2-00-p-2, which started with a Na+ modelled near D244. Red/transparent red/transparent gray: simulation m2-00-f-3 during which a Na+ ion spontaneously entered the binding site and bound in almost the exact same binding pose as in the simulation where the ion was modelled. (C) Cut through the density (same view as (B)) for simulation m2-00-f-3. (D) Cut through the density (same view as (B)) for the simulation M2-00-p-2 (over the part of the trajectory with a Na+ bound). (E) Na+ coordinating residues as see in MD (from trajectory m2-00-p-2, protomer A). The side chain carboxylates of D244 and D215 interacted with the ion together with the backbone carbonyl oxygens for T214 and S240 and typically two water molecules. (F) Na+ coordinating residues (from trajectory m2-00-p-2, protomer B). Only the sidechain carboxylate of D244 contributed to binding together with the backbone carbonyl oxygens of T214 and S240 and typically two water molecules. (G) Membrane lipid density from CG MD simulations. The combined POPC and cholesterol lipid density was contoured at the bulk density to show the extent of interactions between NHE9 (shown as tubes) and lipids. (left) Top view (from extracellular side) (right). Side (in-membrane) view. The circular shape of the membrane patch is an artefact from the rotational superposition of the orthorhombic simulation system prior to density analysis and indicates that the protein was rotating freely in the membrane.
Levels of the Autophagosomal Marker LC3-II Are Increased in Degenerating npc1 Cerebellum.Figure 7(A) Immunoblots of 70 μg of protein resolved by 15% SDS-PAGE show increased levels of LC3-II in 50- and 67-d-old npc1 cerebellar extracts. Two mice of each age and genotype were analyzed, and relative band intensities were quantified. p-Values derived from comparing wild-type and npc1−/− cerebella at each age are 0.3, 0.01, and 0.001 for 22-, 50-, and 67-d-old mice, respectively. (B) Immunoblots of CHO cell extracts resolved by 15% SDS-PAGE do not show increased levels of LC3-II in CT60 cells compared to wild-type CHO-KI cells or CT60 cells stably transfected with NPC1-YFP. No difference is noted with 10 μM U18666A, a drug that mimics npc1 loss of function. Rapamycin treatment (1 μM) of CHO-KI cells demonstrates robust activation of autophagy with increased LC3-II levels.
Xanthine accumulates in age-1; gas-1 mutants.Figure 4A) Volcano plot of lipidomic profiling in gas-1 versus age-1; gas-1 mutants. Red stars: significantly altered lipids in age-1; gas-1 versus gas-1 (5% FDR; n=5). B) NMR-1D targeted metabolic profiling in age-1; gas-1 mutants (normalized to gas-1). Dashed lines indicate fold change cutoff (|log2(fold change)| <1.23). Red bars: not significant, Light blue bars: FDR<10%, |log2(fold change)| <1.23, Dark blue bars: FDR <10%, |log2(fold change)| >1.23. Error bar: mean + SEM, n=7. C) Schematic representation of purine degradation pathway with overlaid Molecule Activity Predictor (MAP) analysis showing increased purine degradation in age-1; gas-1 mutants and the consequent xanthine accumulation. Shades of red and green are proportional to fold change, whereas shades of blue and orange are proportional to the strength of the prediction, as calculated by IPA. D) Left panel: qRT-PCR of xdh-1 (normalized to wt). Error bars: Mean ± SEM, n=6. ****p value< 0.0001, *p value< 0.05, ns= non-significant, Kruskal-Wallis with Dunn's post hoc test. Right: xanthine oxidase activity levels normalized to animal number. Error bars: mean ± SEM, n=2, ns= non-significant, Mann Whitney U test. E) Schematic representations of xanthine and the methylxanthines used in this study. F) Immunoblot analysis of phospho-Akt (S473), pan Akt, phospho-PRAS40 (T246) and total PRAS40 in WT and NDUFA9 KO HAP1 cells treated with 0.2% DMSO (-) or AMP (2 μM), ENPF (2 μM) and PTX (36 μM) (+) for 24 h (actin loading control). (G) Immunoblot analysis of phospho-PKA substrates in WT and NDUFA9 KO HAP1 cells treated as indicated (actin loading control). For all immunoblot panels: numbers correspond to band densitometries across 2-3 biological replicates.
Mitochondrial stressed animals overcome DNA-damage checkpoint.Figure 4Representative fluorescence pictures and quantification (below the fluorescence pictures) of dissected adult gonads relative to the number of mitotic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 and stained with DAPI (blue) and either anti-PARP-1 (red) (A Scale bar: 20µ (A Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and condition Representative fluorescence pictures and quantification (below the fluorescence pictures) of dissected adult gonads relative to the number of mitotic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 and stained with DAPI (blue and eithe anti-RPA-1 (green) and anti-RAD-51 (red) antibodies and treated with gamma radiation (125 Gy) (B) (untreated animals displayed no staining of RPA-1 and RAD-51, not shown) 15µm (B Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and condition Quantification of mitotic cells in wild-type animals fed as (A) and left untreated or treated with gamma radiation 125 G Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and condition Quantification of mitotic cells in wild-type animals fed as (A) and left untreated or treate with 1200 J/m2 UV Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and condition
a) HeLa cells transfected for 5 days with two rounds of control, epsin 1 (eps.figf2(a) HeLa cells transfected for 5 days with two rounds of control, epsin 1 (eps.), clathrin heavy chain (cla.), AP2 or AP1 siRNA were either left untreated or treated for 4 h with bafilomycin A1 (+/- BafA1), after which they were lysed for western blot analysis with anti-LC3 and anti-tubulin antibodies. Note that LC3-I is often very faint compared with LC3-II in HeLa cells under the protein extraction conditions we use. However, this is not a problem because it is advisable to relate LC3-II to tubulin/actin. (b) Ratio of LC3-II to tubulin on knockdown (KD) of control, epsin, clathrin heavy chain, AP2 or AP1, with or without bafilomycin A1, quantified from three independent experiments (two for epsin) and represented in the graph. Asterisk, P 0.01; two asterisks, P 0.001; three asterisks, P 0.0001. (c) HeLa cells stably expressing GFP-mRFP-LC3 (ref. 17) were transfected for 5 days with two rounds of control or clathrin heavy chain siRNA, during which they were either left untreated (UT) or treated with bafilomycin A1 (BafA1) for the last 15 h. Cells were then fixed and analysed on a Cellomics ArrayScan system. Quantification of autophagic vacuoles (AV) or autolysosomes (AL) per cell in the different conditions is shown in the graph. Asterisk, P 0.01; n.s., not significant. n = 2,000 cells. (d) HeLa cells transfected for 4 days with control, clathrin heavy chain, AP1 or AP2 siRNA were grown in Hanks balanced salt solution (HBSS) for 6 h, after which the cells were fixed and immunostained for endogenous LC3. The percentages of HeLa cells with more than 50 LC3 vesicles (marked with arrows) are quantified in the graph. End., endogenous. Three asterisks, P 0.0001. n = 500 cells. All error bars represent s.e.m. Scale bars, 10 μm. Uncropped images of blots are shown in Supplementary Information, Fig. S9.
Figure 4A, B Equal numbers of male and female cynomolgus monkeys were treated with vehicle control (n=4) or escalating doses AT845 (n=6) and followed for approximately 12 weeks. (A) Vector copy number and (B) GAA activity in heart, quadriceps, triceps, diaphragm, and liver. Statistical analysis: mixed-effects analysis with Dunnett's test. Data are presented as box-and-whisker plots with Tukey whiskers that show minimum, median, and maximum values. Asterisks (*) indicate significant differences compared with vehicle control NHPs. *, P<0.05; **, P<0.01.
a) Diagram shows five putative ARE cis-acting elements in.f7(a) Diagram shows five putative ARE cis-acting elements in humanNDP52 promoter region (2,137 bp). Red ellipsoids indicate the putative ARE cis-acting elements. (b) CN1.4 cortical cells and SH-SY5Y cells were transiently transfected with the reporter plasmids with the human NDP52 promoter (2,317 bp or deleted mutants), and luciferase activity was assayed 24 h after transfection. (c) SH-SY5Y cells were co-transfected with the reporter plasmids with human NDP52 promoter (wild or deleted mutants) along with either the mock plasmid (pcDNA3.1) or the plasmid expressing Nrf2, and the luciferase activity was assayed 24 h after transfection. (d) SH-SY5Y cells were transiently co-transfected with the reporter plasmids with human NDP52 promoter or its mutant plasmid in which the putative ARE sequence was site-directly mutated (X), together with the mock plasmid (pcDNA3.1) or the plasmid expressing human Nrf2. The luciferase activity was assayed after 24 h. The induced luciferase activity with co-expression with Nrf2 was not observed in a mutant NDP52 promoter in which all three ARE sites (−2,057, −1,023 and −94) were site-directly mutated, indicating these three sites of five ARE cis-acting elements are functional. Data shown are mean±s.d. of three independent experiments and were analysed using Student's t-test. (*P0.05; **P0.01; ***P0.001).
AtNTR1 interacts with U6 and U1 snRNA and co‐localise with PolIIElectrophoresis of RT–PCR products showing interactions of AtNTR1 with selected snRNA targets detected by RIP.Figure 2A Electrophoresis of RT-PCR products showing interactions of AtNTR1 with selected snRNA targets detected by RIP. The level of transcripts co‐precipitated from transgenic plants expressing ANTR1‐GFP (IP+) or wild‐type plants (IP−) using anti‐GFP antibody was measured by RT-PCR normalised to the inputs. To control the amplification from gDNA, controls without reverse transcriptase (RT) were performed. U3 snRNA and 18S rRNA were used as negative controls for interaction. B Fluorescent immunostaining of nuclei showing the co‐localisation of AtNTR1 with SC35, total PolII, Ser5‐phosphorylated PolII (PolIIA) or Ser2‐phosphorylated PolII (PolIIO). AtNTR1 was detected using an antibody raised against AtNTR1 peptide. Scale bar represents 2.5 μm.
Figure 3A Whole-endocrine gland imaging of young and aged adrenal glands immunolabelled with α-SMA and Emcn acquired on a light-sheet microscope. Asterisks indicate higher magnifications of regions in adrenal glands from young and aged mice. Combined box and whiskers, and scatter plots with quantifications of vessel density and numbers of arteries. B Whole-endocrine gland light-sheet imaging-based 3D images of young and aged ovaries stained with α-SMA and Emcn. Asterisks represent higher magnifications of regions in ovaries from young and aged mice. Combo plots show quantifications of vessel density and numbers of arteries in young and aged ovary. C 3D images of whole cleared young and aged testes stained with α-SMA and Emcn. Asterisks indicate higher magnifications of indicated regions. Combo plots show quantifications of vessel density and numbers of arteries. D Light-sheet imaging of whole cleared young and aged pancreases immunolabelled with α-SMA and Emcn. Asterisks indicate higher magnifications of regions. Combo plots with quantifications of vessel density and numbers of arteries in young and aged pancreas. E 3D images acquired on a light sheet microscope of whole cleared young and aged thyroid glands stained with α-SMA and Emcn. Asterisks indicate higher magnifications of regions. Combo plots show quantifications of vessel density and numbers of arteries in young and aged thyroid glands. 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.
a) THPN inhibits the formation of tumors in the liver and lung in a TR3-dependent manner.figf6(a) THPN inhibits the formation of tumors in the liver and lung in a TR3-dependent manner. B16 cells transfected with either control shRNA, TR3 shRNA or ATG7 shRNA (5 × 105) were separately injected into the lateral tail vein of 6-week-old C57BL/6J mice (n = 7). THPN were administered 3 d after inoculation. The tumors formed in the lung and liver are indicated (top; scale bars, 5 mm) and were counted (bottom). Ctrl, control. (b) Crossed Tyr:NRasQ61K;Ink4a/Arf−/−;TR3+/+ and Tyr:NRasQ61K;Ink4a/Arf−/−;TR3−/− mice (n = 12) were treated topically with DMBA to initiate cutaneous melanocytic neoplasms, and mice were treated with THPN as described in Online Methods. The representative images of dorsal melanomas (top) and the tumor numbers (bottom) are presented. (c) The survival curves of crossed Tyr:NRasQ61K;Ink4a/Arf−/−;TR3+/+ and Tyr:NRasQ61K;Ink4a/Arf−/−;TR3−/− mice are shown (n > 16). (d) A signaling pathway of THPN-induced and TR3-mediated autophagic induction in the inhibition of melanoma growth. All of the data are presented as the mean ± s.e.m. of three independent experiments. *P 0.05, **P 0.01; NS, not significant.
Effects of FGF2 on apoptosis and cell cycle progression in BET inhibitor-treated UM cells.Figure 4
TORC1 interacts physically with translation preinitiation complexes.Figure 4(a) Binding of eIF4G to the Rag GTPases detected by co-immunoprecipitation from Kc167 cells in absence or presence of the chemical cross-linker DSP. Representative of >3 biological replicates. (b) Binding between eIF4G and the Rag GTPase complex is mainly mediated by RagC, detected by expressing and immunoprecipitating either RagA or RagC alone. Representative of two biological replicates. (c) Interaction between eIF4A and Raptor detected via co-immunoprecipitation of epitope-tagged proteins. Proteins were cross-linked with DSP prior to lysis and immunoprecipitation. Representative of 3 biological replicates. (d-d'') eIF4A and Raptor form a complex, detected by proximity ligation assay (PLA). Specificity of the PLA signal was controlled by knocking down either eIF4A or Raptor in Kc167. (d) Representative images. Cells outlined in white. (d') Quantification of number of PLA spots per cell. (n>200. ***p<0.0001, ANOVA1, Dunnett's multiple comparisons test, was performed on log-transformed data, box = +/- 1 quartile, whiskers = max/min). (d'') Immunoblotting to detect efficiency of eIF4A and Raptor knockdowns. (e-f) The eIF4A-Raptor interaction does not take place on lysosomes (e) or the endoplasmic reticulum (ER) (f). Kc167 cells were either incubated with 100 µg/mL Dextran for 1 hour, washed and incubated for 14 hours in normal growth medium to label late endosomes and lysosomes (e), or transfected to express an ER-resident GFP (f). Amino acid removal was for 45 min. Pearson's correlation coefficients for eIF4A-Raptor PLA and Dextran in panel (e) are 0.09 and 0.08 for +aa and -LIVASTQP respectively (calculated for all cells in the acquired fields, which were chosen randomly, n=40). Scale bars: 5µm.