EMBO/sd-character-level-ner huggingface dataset:
Figure 5A Lysates from MEF-WT or MEF-Cuedc2-/- cells treated with 20μM cycloheximide (CHX) for the indicated times were subjected to immunoblotting (left). Relative GPX1 levels were quantified by densitometry (right). B MEF-Cuedc2-/- cells were transfected with increasing amounts of FLAG-CUEDC2 (mouse) plasmids (1 μg and 2 μg). At 24 hours after transfection, the cells were treated with the proteasome inhibitor MG-132 (10 μM). Cells were cultured for additional 6 hours and subjected to immunoblotting. C HEK293T cells were transfected with the FLAG-GPX1, HA-CUEDC2, and Myc-ubiquitin plasmids as indicated and treated with MG-132 (10 µM) for 6 hours before harvest. Cell lysates were immunoprecipitated (IP) with anti-FLAG (M2). The immunoprecipitates were analyzed by western blot using an anti-GPX1 antibody. Whole-cell lysates (WCL) were analyzed by Western blots with anti-FLAG or anti-HA antibody to determine the protein of Flag-GPX1 and HA-CUEDC2. D The heart tissue lysates from wide-type and Cuedc2-/- mice were incubated with IgG or anti-GPX1 antibody, and then captured on protein G-Sepharose beads. The immunoprecipitates were analyzed by immunoblotting with an anti-ubiquitin antibody. E HEK293T cells were transfected with the Flag-CUEDC2 or Flag-CUEDC2△CUE (deletion of the CUE domain of CUEDC2), and treated with MG-132 (10 µM) for 6 hours before harvest. Cell lysates were immunoprecipitated (IP) with anti-Flag (M2). The immunoprecipitates were analyzed by western blot using an anti-GPX1 antibody. Whole-cell lysates (WCL) were analyzed by Western blots with anti-Flag or anti-GPX1 antibody to determine the protein of Flag-CUEDC2 and GPX1. v: vector.
Figure 7HEK293 cells were transfected with siCTL, siDDX5, or siXRN2. The gene location and qPCR amplification region are shown at the top of each panel. The identified R-loop peaks were extracted from R-loop database (R-loop DB) for each gene region. R-loop: DRIP denotes anti-S9.6 DRIP-qPCR and Pol II ChIP denotes RNA pol II ChIP. The bar graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001. B, E, H, S and X denote the location of the BsrGI, EcoRI, HindIII, SspI, and XbaI.
A model of the mitochondrion"s life cycle that integrates mitochondrial dynamics and turnover.Figure 9A model of the mitochondrion's life cycle that integrates mitochondrial dynamics and turnover. The mitochondrion cyclically shifts between a post fusion state (network) and a post fission state (solitary). Fusion is brief and triggers fission. Following a fission event, the daughter mitochondrion may either maintain intact membrane potential (red line) or depolarize (green line). If it depolarizes, it is unlikely to proceed to a subsequent fusion, unless it re‐polarizes. After being depolarized and solitary for a few hours, the mitochondrion is removed by autophagy.
Figure 7Mitochondrial translation products were pulse-labelled in control 143B cells (left panel), COX1∆ (middle panel) and COX2∆ (right panel) cybrids with [35S]-methionine for 30 minutes in the presence of anisomycin to inhibit cytosolic translation. For chase, cells were washed and incubated with fresh culture medium for the indicated time points (T0-T24 hours). Radiolabeled mitochondrial proteins (indicated on the right) were separated by 2D-BN/SDS-PAGE and visualized by autoradiography. A enlarged section of the SCs region at T24 (delimited by a square) shows the co-migration of COX1 with SC I+III2plus (black arrow) in the COX2∆ cybrids relative to the position of canonical SC I+III2 (gray arrow). The identities of MRC complexes and SCs (bottom) are as in Figure 1. Molecular weight markers are indicated on the left.
Figure 7Rab39a expression in splenocyte subsets was measured using an FDG assay. Shown are relative FDG fluorescence of Rab39a KO cells/WT background. Summary of two independent experiments. Splenic DCs were expanded in Rab39a KO and WT mice by injection of B16-Flt3L tumor. 10 days post tumor implantation, DCIR2+ (Cd8α-) or XCR1+ (Cd8α+) dendritic cells were isolated from spleens via magnetic positive selection. 5 x 104 cells were plated in a 96 well plate and incubated with the indicated amounts of Ova-Fe and 1:1 of Reporter CD8 or CD4 T-cells overnight before measuring luciferase activity. Error bars indicate SD of 2 replicate wells. Shown are representative graphs of 3 independent experiments. * p < 0.05 using two-way ANOVA. CD11b+ CD11c+ dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection. Cells were fed with 3 µm biotin-ova-magnetic beads for 4 hours. Phagosomes were extracted, permeabilized and stained for the indicated proteins. For ROS measurements, phagocytosis was performed with 5 µM CellROX and the phagosomes were not permeabilized. For analysis, the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of target (dox/no dox) was calculated. Shown are three independent experiments combined. Error bars show SD between experiments. p values were calculated using two-tailed ratio-paired Student's t-test. CD11b+ CD11c+ dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection. Cells were fed with 3 µm magnetic beads conjugated to pHrodo. After 4 hours, cells were harvested for flow cytometry to measure fluorescence of eaten beads due to phagosomal pH. For analysis, the background fluorescence of unstained beads was subtracted from the resulting gMFI and the fold change (dox/no dox) was calculated. Shown are three independent experiments with error bars indicating the SD. p values were calculated using two-tailed ratio-paired Student's t-test. CD11b+ CD11c+ dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection. Cells were fed with 3 µm magnetic beads conjugated to Alexa fluor dye. After 4 hours, cells were harvested and resuspended in 1 mg/ml Trypan blue to quench fluorescence of uneaten beads. Cells were run for flow cytometry to measure phagocytosis. The ratio of KO cells with beads / WT cells with beads was then obtained. Graphs show three independent experiments and p values were calculated using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments. Rab39a KO and WT mice were injected intravenously with 1 x 105 A20-Ova cells. The next day, CFSE labeled OTI-splenocytes were injected i.v.. 3 days post OT-I transfer, spleens were harvested and assayed for OT-I proliferation. Three independent experiments (WT vs KO) were performed. To combine the data, the OT-I proliferation in each individual mouse was normalized to the average proliferation of the WT controls in that experiment. Error bars show SD between individual mice and p value was derived from two-tailed student's t-test. n = 14 (WT) or 9 (KO).
Cardiac LXRα overexpression enhances myocardial glucose uptake.Figure 4Classification of genes differentially expressed in LXRα-Tg hearts (see Supplementary Fig S3). Representative 18F-FDG images with microPET in the coronal and axial planes in Wt and LXRα-Tg mice 5 weeks post-TAC. Myocardial FDG uptake measured as standard uptake value (SUV); n = 4-6/group. *P = 0.04 versus Wt sham, **P = 0.02 versus LXRα-Tg sham, #P = 0.03, ##P = 0.01. Quantification of myocardial glycogen content shows no significant differences; n = 5 per sham group, n = 7 per TAC group. GLUT protein expression in LV tissue normalized to GAPDH; n = 6/group. Glut1: *P = 0.01 versus LXRα-Tg sham, #P = 0.03; Glut4: *P = 0.03 versus LXRα-Tg sham, #P = 0.01.
Figure 2MHC-II presentation of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-Fe. The cells were then exposed to MF2.2D9-Luc Reporter CD4 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 vs negative control β2m using two-way ANOVA. None of the bead concentrations are p < 0.05 for Rab39a siRNA vs β2m. Representative plot of 3 independent experiments DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 cells were then fed with Ova-Fe and exposed to reporter T cells as in (A). Error bars show SD of duplicate wells. None of the bead concentrations are p < 0.05 for Rab39a siRNA vs β2m using two-way ANOVA. Representative plot of 3 independent experiments MHC-I Presentation of SIINFEKL-H2-Kb complexes on siRNA treated DC3.2 NS-Ova (C) or DC3.2 UbS8L (D) cells. 48 hours after siRNA transfection, the indicated amounts of dox was added to the cells to induce Ova expression. After 2 hours at 37oC, RF33.70-Luc CD8 T cells were added along with Brefeldin A (Golgiplug) to a final concentration of 1:1000. The cells were incubated overnight for reporter T cell luciferase expression. Error bars show SD of >3 replicate wells. * p < 0.05 vs negative control I-Ab using two-way ANOVA. None of the dox concentrations are p < 0.05 for Rab39a siRNA vs I-Ab. Representative plot of 3 independent experiments Flow cytometric analysis of surface H2-Kb and I-Ab levels of siRNA treated DC3.2R cells. Shown are results after 48 hours of knockdown. Representative plot of 3 independent experiments
Figure 3(A) Animal protocol. (B) Duodenal CRAMP determination by ELISA (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (C) Changes in body weight relative to starting weight during 6 weeks (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. (D) Left, representative images of duodenal damage by H&E staining. Scale bar: 200 μm. Right, graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (E) Western blot and densitometry analyses of duodenal tight junction proteins (claudin-1, occludin, ZO-1 and ZO-2; n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (F) Western blot and densitometry analysis of duodenal zonulin (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (G) Intestinal permeability was assessed by measuring FITC-dextran (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.
Figure 8Schematic representation of the β-actin locus and the location of the genomic PCR primers used for ChIP-qPCR and DRIP-qPCR. Prom (promoter), RB (region B), poly(A) and RD (region D) indicate the regions analyzed by qPCR. HEK293 cells were transfected with siCTL or siDDX5 and XRN2 ChIP assays were performed. The XRN2 ChIP signal was normalized to the Pol II ChIP signal. WCE were immunoblotted with anti-DDX5 and α-tubulin antibodies, the latter was used as a loading control. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01. HEK293 cells were transfected with siCTL, siDDX5 or siXRN2. The cells were subjected to Western blotting, RNA Pol II ChIP and DRIP analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001. HEK293 cells were transfected with siCTL or siDDX5. The next day, the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector (-), expression vectors encoding Flag-DDX5 (WT), or Flag-DDX5-RK (RK). The cells were subjected to Western blotting to visualize Flag-DDX WT and RK expression. Pol II ChIP and DRIP analysis of β-actin gene were performed. For the ChIP analysis, the Y-axis shows the signal-to-noise ratio of RNA Pol II IP relative to control IgG IP. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001. HEK293 cells were transfected with siCTL or siPRMT5. The cells were subjected to Western blotting, RNA Pol II ChIP and DRIP analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01. HEK293 cells were transfected with empty plasmid (-), wild type Flag-DDX5 (WT) or its RK mutant (RK) and subjected to western blot and Flag-DDX5 ChIP-qPCR analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001; n.s. not significant.
Figure 1A Cartoon of the morphogen hypothesis: a spatially varying concentration of signaling molecule can result in precise readout of positional information. B Outline of models considered. Morphogen RNA (grays) and protein (green dots) distribution are shown in early (top) and cycle 14 (middle) rows for each model considered. The magnitude of protein movement is represented by length of green arrows. Degradation of protein/RNA is represented by green/gray arrows. Bottom row shows a schematic of morphogen RNA and protein concentration profiles in cycle 14. C Normalized Bcd concentration profiles for the models considered in b at time=2.5 hours. Inset: same on log scale. D Data points correspond to mean output of stochastic Monte Carlo simulation results for the average protein age as function of position, 2.5 hours after initiation Colored lines correspond to theoretical predictions for protein age in each model showing solutions over larger parameter space. Solid line represents mean solution and dashed lines represent 1s.d. Parameter range described in text. SDD: synthesis, diffusion, degradation model, NucSh: nuclear shuttling model, RNA-grad: RNA gradient model, and RNA-diff: RNA diffusion model.
Vaccination with attenuated CHIKV protects mice against WT O'nyong nyong virus (ONNV).Figure 7Three-week-old WT C57BL/6 mice were infected with 1E6 PFU of WT-ONNV at the metatarsal region of the footpad. (A) Viremia progression in ONNV-infected mice were monitored over 2 weeks (n=5). Viremia is detected with ONNV nsP1 probe via qRT-PCR. Three-week-old WT C57BL/6 mice were infected with 1E6 PFU of WT-ONNV at the metatarsal region of the footpad. (B) joint inflammation in ONNV-infected mice were monitored over 2 weeks (n=5). Three-week-old WT C57BL/6 mice were vaccinated subcutaneously with 1E6 PFU of WT-CHIKV or attenuated CHIKV (RH) at the metatarsal region of the footpad. Challenge with 1E6 PFU of WT-ONNV was performed via the same route at 3 months post-vaccination. Viremia progression of the mice were monitored over 2 weeks (n=5 per group). Viremia is detected with ONNV nsP1 probe via qRT-PCR. D) Three-week-old WT C57BL/6 mice were vaccinated subcutaneously with 1E6 PFU of WT-CHIKV or attenuated CHIKV (RH) at the metatarsal region of the footpad. Challenge with 1E6 PFU of WT-ONNV was performed via the same route at 3 months post-vaccination. (D) joint inflammation of the mice were monitored over 2 weeks (n=5 per group).
Figure 6(a) Reaction schemes for experiments in (b) and (c). (b) Immunoblots of reactions performed as in (a) part i (lanes 1-3) or ii (lanes 4-6). (c) Reactions in i and ii were performed as described in (a) parts i and ii, respectively.
Mutant analysis allows for differentiation between vacuolar delivery by Cvt vesicles and autophagosomes.Figure 5Mutant analysis allows for differentiation between vacuolar delivery by Cvt vesicles and autophagosomes. (A) SEY6210 (wild-type), cvt3, and cvt7 yeast were grown in SD to 1 OD600 U/ml and transferred to SD (-N). Aliquots were collected after 0 h (+) and 4 h (−) incubation and subjected to immunoblotting. The positions of precursor (pro) and mature (m) API are indicated. (B) cvt3 and cvt7 cells were pulse labeled for 10 min. After addition of cold cysteine and methionine, the cells were harvested, washed, and resuspended in either SD or SD(-N) and chased for the indicated times. API was recovered by immunoprecipitation, resolved on SDS polyacrylamide gels, and quantified using a phosphorimager (STORM; Molecular Dynamics, Sunnyvale, CA).
Viability of respiratory deficient cells during nitrogen starvation.Figure 5(A) WT and atg1Δ cells were transferred to non-buffered SD-N medium for the indicated time. Cell viability was determined by phloxine B staining. Nitrogen-starved cells were plated onto YEPD agar and overlaid with TTC agar to examine the respiratory competency of formed colonies. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. (B) WT, atg1Δ, rho0, and rho0 atg1Δ cells grown in YEPD medium were transferred to SD-N with or without 10 mM NAC for the indicated time. In the presence of NAC, medium pH was adjusted by using KOH. Cell viability and medium pH were examined by phloxine B staining and pH meter, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.
Rb−/− myoblasts transiently differentiate to form short myotubes that do not twitch, express autophagy markers, and slowly degenerate.Figure 1.(A) Western blot analysis for pRb in DM-2 cultures. (B) Immunostaining for MHC in DM-3 and DM-6 differentiating myotubes. Nuclei were stained with DAPI (blue). (C) Mean number of myotubes on the indicated days after differentiation. Each time point is mean ± SD of six fields from six independent experiments. (D) TUNEL staining. Arrowheads indicate TUNEL-positive nuclei (green). (E) LC3-RFP expression in control (ctrl) and Rb−/− myotubes. Insets show nuclear DAPI stain (blue). (F) Representative Western blot (n = 3) for LC3. Chloroquine was added 12 h before cell harvest. (G) Representative Western blot for LC3 in E16.5 control and mgRb:Rb−/− muscles. (H) Quantification of LC3-II/tubulin ratio in E16.5 mgRb:Rb−/− skeletal muscles relative to control as depicted in G (mean ± SD; n = 3). *, P = 0.016 by Student's t test. (I) Representative Western blot for LC3-I and LC3-II expression in whole cell lysates from the indicated myotubes transduced with Ad.GFP or Ad.RbΔK11. (J) Quantification of LC3-II/tubulin ratio by Western blots as shown in I. Data represent mean ± SD (n = 7). *, P = 0.047; **, P = 0.005 by analysis of variance.
Figure 5A) Principal component analysis with the 1000 most variable genes from microglial cells isolated from CX3CR1GFP/+ and CX3CR1GFP/+;Sfrp1-/- mouse brains three days after saline or LPS intra-cerebro-ventricular infusion. The analysis depicts sample clusterization by genotype and treatment. B, C) Volcano plots of differential gene expression from CX3CR1GFP/+ (B) or CX3CR1GFP/+;Sfrp1-/- (C) microglial cell in response to LPS. Data are represented as Log2 Fold Change vs -Log10 adjusted p-value by the Wald test corrected for multiple comparisons by the Benjamini and Hochberg method. Blue vertical lines represent an increase of 75 and 400% in the expression levels respectively. Green horizontal lines represent a 0.05 or 0.01 adjusted p-value of statistical significance respectively. Genes with an expression change higher than 75% and an adjusted p-value lower than 0.05 are highlighted in dark red (+FC) and dark blue (-FC). Genes with an expression change higher than 400% and an adjusted p-value lower than 0.01 are coloured in light red (+FC) and light blue (-FC). D) Scatterplot of differential expressed genes in CX3CR1GFP/+ and CX3CR1GFP/+;Sfrp1-/- microglia; the red line represents linear regression (slope 0.704014 p-value: < 2.2e-16 by F-test followed by Bonferroni's correction) of the differential expression, indicating response attenuation. Coloured dots represent response which are exclusive for each genotype as represented in E. E) Venn diagram showing the extent of differential gene expression in response to LPS in CX3CR1GFP/+ or CX3CR1GFP/+;Sfrp1-/- microglial cells. Fold change (75%) and adjusted p-value < 0.05 cut-off by the Wald test corrected for multiple comparisons by the Benjamini and Hochberg method. F) Heatmap showing fold changes of regularized log transformed gene-level RNA-seq counts with hierarchical clustering of samples and differentially expressed genes in response to LPS in CX3CR1GFP/+ or CX3CR1GFP/+;Sfrp1-/- microglial cells.
Figure 3Dimer of full-length cGMP-bound Bd1971; one monomer coloured individually by subdomain, the opposing monomer coloured white. Ligand represented in stick form, EAL active site Mg2+ as green sphere. View from top of that in (A), detailing EAL region of dimer interface. View from opposing side, detailing C-helix dimer interface at cNMP domain. The Bd1971 EAL dimerization interface (purple, white) matches that of a canonical, active dimer from the well-characterized T. denitrificans EAL (tan, PDB 2r6o); the catalytic Asp pairs of the two enzymes adopt a similar conformation (stick form, D306/307 from Bd1971, D646/647 from 2r6o). Structure of the cyclic-di-GMP bound complex of Bd1971 (ligand C represented in yellow, bound Ca2+ ions as green spheres). Residues R182, R330 and Y385 display the largest shift between the apoenzyme (tan) and bound (blue) states, R182 complexing the cyclic-di-GMP phosphate, and Y385 stacking with a guanine base.
Associations between components of the retinoid signaling pathway and the phenotype in breast cancer cell lines.Figure 4Associations between components of the retinoid signaling pathway and the phenotype in breast cancer cell linesThe figure illustrates the associations between the indicated retinoid receptors/binding proteins and the Luminal versus Basal phenotype (left panels), ER positivity versus ER negativity (middle panels) as well as HER2 positivity versus HER2 negativity (right panels). The gene-expression microarray and RNA-seq data refer to 42 and 40 breast cancer cell lines, respectively. The P-values of the indicated comparisons after Student's t-test are shown in red. L (red) = Luminal cell lines; B (blue) = Basal cell lines; ER+ (red) = ER-positive cell lines; ER− (red) = ER-negative cell lines; ER− (blue) = ER-negative Luminal cell lines; H+ (red) = HER2-positive cell lines; H− (red) = HER2-negative cell lines; H+ (blue) = HER2-positive Luminal cell lines; H− (blue) = HER2-negative Luminal cell lines. fpkm = fragments per kilobase of exon per million fragments mapped.
Figure 4B. SecB protects proOmpA* from aggregation. proOmpA*1 (5 μM) was incubated (50 μl; Buffer C; 37°C; 60 min) alone or in the presence of the indicated molar excess of SecB4, and proOmpA* in the soluble fraction (after centrifugation) was analyzed by SDS-PAGE (as in Fig. 2C). Lane 2: 2.84 μg proOmpA*. Representative experiment shown; n= 4 biological replicates  C. SecB physically interacts with proOmpA. proOmpA1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 4°C; 60 min), with the indicated molar excess of SecB4 (filled arrow), analyzed by native-PAGE as in Fig. 2A, and stained as indicated. brace: higher order proOmpA species. Representative experiment shown; n= 5 biological replicates