EMBO/sd-character-level-ner huggingface dataset:
a) USP33 does not affect RALB activity.figf3(a) USP33 does not affect RALB activity. The activity of RALB was determined in HEK293T cells expressing GFP shRNA or USP33 shRNA and empty vector (V) or HA-tagged USP33 by RALBP1-RBD binding assay. (b) USP33 modulates interaction between RALB and SEC5. Flag-tagged RALB was overexpressed in HEK293T cells expressing GFP shRNA, USP33 shRNA, empty vector (V) or HA-tagged USP33. The indicated proteins were then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-Flag or anti-SEC5 antibodies. (c) USP33 regulates RALB binding to EXO84. Flag-tagged RALB was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells expressing the indicated constructs. The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti-HA or anti-EXO84 antibody. (d) Activated RALB co-localizes with both EXO84 and SEC5. At 24 h after co-transfection with RALB-G23V-EGFP and HA-EXO84, HeLa cells were immunostained with anti-HA and anti-SEC5 antibodies. Scale bars, 10 μm. The outlined areas are shown at higher magnification in the bottom panels. (e) RALB ubiquitylation controls interaction between EXO84 and SEC5. Flag-tagged SEC5 was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing RALB-G23V or RALB-G23V-K47R. The presence of EXO84 in SEC5 complexes was analysed by immunoblotting using anti-EXO84 antibody. (f) USP33 regulates EXO84-SEC5 complex formation. Flag-tagged SEC5 was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells expressing the indicated constructs followed by immunoblotting using anti-EXO84 antibody. (g) Suppression of SEC5 affects RALB binding to EXO84. HA-tagged RALB-G23V was immunoprecipitated from HEK293T cells expressing the indicated constructs. The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti-EXO84 antibody. For a-c,f,g immunoblotting using antibodies specific for USP33, anti-Flag (M2) or anti-HA determined the levels of USP33 suppression or overexpression. Immunoblot analysis was performed using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S7.
Significant motifs among sites undergoing acetylation.Figure 6.Significant motifs among sites undergoing acetylation. (A) Hierarchical clustering of the organellar distributions of sites whose acetylation was increased by >1.5-fold in response to resveratrol or spermidine, at least in one organellar fraction. (B) Consensus acetylation motifs identified upon resveratrol (left) or spermidine (right) treatment are depicted using the MotifX algorithm (Schwartz and Gygi, 2005). Among sites that were hyperacetylated in response to both agents, the K(F/Y) motif is significantly enriched when tested against the whole proteome (P < 0.00001). (C) When testing against the largest acetylation site dataset from Choudhary et al. (2009) (acetylation background dataset [ABD]), the SxK motif is significant (P < 0.0001) for sites whose acetylation increased upon spermidine treatment. No general consensus motifs were found for sites whose acetylation increased in response to both agents.
A speculative model for ectopic formation of structures related to centrioles upon RBM14 depletion.Figure 7
Additive protection of zinc and dexamethasone against TNF-induced lethal inflammation.Figure 7(A) C57BL/6J mice received 25 mM ZnSO4 or control water for 7 days. Mice were injected i.p. with 100, 200 or 500 µg dexamethasone (Dex, rapidexon), solved in sterile PBS, 30 min before a 50 µg TNF/20 g bodyweight i.p. challenge. STAT1+/+ and STAT1-/- mice were challenged with equal TNF dose, as controls. No mice died later than 120 h after TNF injection. Data show combined results of 2 experiments. Mice (N=4 per group) treated as in (A) and 6h after challenge, ileum was taken and RT-qPCR performed to study gene expression of cell death ISRE genes Mlkl and Zbp1 Mice (N=4 per group) treated as in (A) and 6h after challenge, ileum was taken and RT-qPCR performed to study gene expression of cell death ISRE genes Stat1. (E) Hypothesis of the mechanism of interference of zinc in TNF-induced cell death in ileum (Paneth) cells. See text for more details.
Modulation of Sae2 recruitment and activity by Sir3 A.Figure 7A. Relative fold enrichment of Sae2 at 0.2 kb from I-SceI site was evaluated by qPCR after ChIP with anti-GFP antibodies. The error bars indicate the variation between at least three biological replicas (SEM). B. Relative fold enrichment of Sae2 at 0.2 kb from TEL6R after DSB induction at the LYS2 I-SceI site was evaluated by qPCR after ChIP with anti-GFP antibodies. The error bars indicate the variation between at least three biological replicas (SEM). Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns). C. Representative images of sae2-E267E mutant cells overexpressing or not full-length Sir3. Scale bars are 2 μm. D. Survival frequencies after DSB induction at LYS2 locus in sae2-E267E mutant cells overexpressing or not full-length Sir3 (oeSir3). Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).
a) Dose-dependent binding of IL-17A to fungal Crh11p (red circle) and Gas1p (light blue circle) proteins, but not to the fungal polysaccharides β-1,3-D-glucan (open circle), mannan (blue circle) and zymosan (grey circle).f4(a) Dose-dependent binding of IL-17A to fungal Crh11p (red circle) and Gas1p (light blue circle) proteins, but not to the fungal polysaccharides β-1,3-D-glucan (open circle), mannan (blue circle) and zymosan (grey circle). The lack of binding of IL-17E to Crh11p (orange circle) serves as a negative control. The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay. Data are mean±s.d., n=3. P0.001, Crh11p and Gas1p without versus with IL-17A (one-way analysis of variance Bonferroni post-test). (b) Co-immunoprecipitation assay of fungal Crh11p and murine IL-17A proteins. Immunoprecipitates were probed with antibodies to the corresponding antigens and secondary HRP-conjugated antibody. C, irrelevant antibody. (c) Failure of IL-17A to bind to wild-type (red square) or mutant C. albicans strains in which the three genes of the CRH family (UTR2, open light blue, CRH1, open black and CRH12, open blue square) had been deleted, and restoration of binding by CRH1 (black square) and CRH12 (blue square), but not UTR2 (light blue square), reconstitution. Data are mean±s.d. (n=3). P0.001 for red square, open light blue, black square and blue square (one-way analysis of variance Bonferroni post-test). (d,e) Cellular localization of bound IL-17A. Electron micrographs of ultrafine sections of C. albicans MKY 378 wild-type (d) or the crh11Δ mutant (e) incubated with biotinylated IL-17A and streptavidin gold-conjugated particles. Arrows indicate immunogold particles. Scale bars, 200 nm.
Figure 3A, B SAXS profiles of the nuclesomal arrays in 0 (TE), 1, 2.5 mM MgCl2 (A) and 5, 10 mM MgCl2 (B) are shown as plots of log(I × S2) vs. 1/S (I, intensity; S, scattering vector (1/nm)). C Two types of nucleosome positioning: face-to-face, at ~6-nm spacing, and edge-to-edge, at ~11-nm spacing. The image was made based on the structural information published in (Luger et al., 1997).D The modeled scattering profile of an extended dinucleosome structure based on its atomic coordinate (for details, see Materials and Methods). Note that the modeled profile is similar to that of the nucleosomal arrays in 0 mM MgCl2 (A).E Two structural models of 12-mer 30-nm fibers: solenoid (left) and zigzag (right) as a top and side view. The models were constructed using MolScript (Kraulis, 1991).F The scattering profiles of the solenoid (red line) and zigzag (blue line) 30-nm fibers were made from their atomic coordinates computationally. Note that the 30-40 nm peak is prominent in both fiber models.
Fig.Figure 3Representative flow cytometric showing the specific depletion efficiency of ATM subsets from eWAT of CD169-DTR ND- (A) or HFD-treated (16 weeks) (B) mice. A representative analysis of three independent experiments is shown. eWAT was collected from lean (C) and obese (HFD 16 weeks) (D) WT or CD169-DTR mice after 12 days DT-mediated ATM depletion. Representative pictures of dissected eWAT are shown on the left, the corresponding eWAT weights expressed as mean ± SD of more than 12 samples are shown in the right, with statistical significance calculated using the Student's t-test (**P < 0.01 and ****P < 0.0001). The zoomed-in area shows clear, oily lipid accumulation in the absence of ATMs (lower left part). Adipocyte hypertrophy is visible in the absence of ATMs. Representative images of hematoxylin and eosin staining (H&E) (E) and corresponding adipocyte diameters (F) of lean and obese (HFD 16 weeks) eWAT from WT or CD169-DTR mice treated for 7 or 12 days with DT. Scale bar, 50 μm. Each group comprised 3-5 mice. Statistical significance was determined using one-way ANOVA test. **P < 0.01; ****P < 0.0001; ns: no significant difference. The box and whisker plots show the median value and 10-90 percentiles of adipocyte diameters. Relative expression of adipokines (adiponectin, leptin, and resistin) in eWAT obtained from WT or CD169-DTR mice, as measured by qPCR after 4, 7, and 12 days DT treatment. Black circles, ND-fed mice; white circles, HFD-fed (16 weeks) mice (n ≥ 5 mice per group, mean ± SD).
Widespread modulation of epigenetic landscape for differentiated organoids is driven by Hnf4g.Figure 3 (A) Heatmaps showing epigenome dynamics on peaks with significant changes in DNA accessibility and histone modifications between CV, ENR and EN organoids. Heatmaps are split on their location near a transcription start site (<500 bp from TSS, top heatmaps) or other location. Heatmaps are further subdivided based on the dataset it was found significantly changing in (shown at the left). Shown p values are corrected for multiple testing hypotheses with the BH method (B) Clustered genomic regions from A are shown. Regions were associated with its single closest TSS and corresponding mRNA expression changes is shown in the most left heatmaps. The best match score for the HNF4G motif is shown in the middle heatmap. Hnf4g binding is shown in the most right heatmaps. (A,B) Relative changes between different organoids are shown as log2 row mean subtractions (RMS) where the signal is compared to the row mean of all samples  (C) Scatterplot with the transcription factors that are most likely to explain the dynamics shown in A. The x axis is the linear correlation between the best motif score and the log2 fold change EN over ENR under each significantly changing peak for a given dataset as shown in A. The y axis is the feature importance of the motif in a random forest model in percentage increase in mean squared error (MSE), where motifs that are important for the model to accurately predict the given fold changes get a high error increase. Different datasets are color coded. For motifs with more than one transcription factors associated to it, we show the transcription factor name with the most significant change in protein expression. The vertical line over each dot represents two times the standard deviation of the MSE  (D) Heatmaps showing change in protein (left) and mRNA (right) expression of selected transcription factors, compared to ENR organoids
UBE2QL1 depletion affects ubiquitination of damaged lysosomes.Figure 2A HeLa cells were transfected with different UBE2QL1 siRNAs for 60 h, and LLOMe or control-treated (untreated) for 3 h. Cells were fixed and immuno-stained for LAMP1 along with antibodies specific for K63 or K48-linked ubiquitin chains as indicated and imaged with a laser scanning confocal microscope. Note the strong reduction of damage-induced K48 ubiquitination and decreased intensity of the K63 signal. Scale bar: 20 µm. B, C Automated image quantification of (A). Shown are percentages of K48 or K63-positive LAMP1 vesicles per cell. Graphs represent data from three independent experiments with ≥ 70 cells per condition (mean ± SD). **P<0.01; ***P<0.001; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).
ERp18 Modulates Trafficking and Cleavage of ATF6α Under no stress (light green shading), BiP binds to the lumenal domain of disulfide bonded monomer and dimer ATF6α, preventing ER exit (Step 1).Figure 9Under no stress (light green shading), BiP binds to the lumenal domain of disulfide bonded monomer and dimer ATF6α, preventing ER exit (Step 1). In step 2, under conditions of ER stress (light red shading), ERp18 or other PDI family members could reduce ATF6α disulfides resulting in the release of BiP from ATF6α. ERp18 binds, possibly before or after BiP release, and regulates ER exit. Reduced ATF6α is subsequently packaged into COPII vesicles and transported to the Golgi apparatus (step 3). The regulated binding and release of chaperones and oxidoreductases ensures that only reduced ATF6α, which is the optimal substrate for S1P, is transported to the Golgi. Sequential cleavage by S1P and then S2P, leads to membrane release and production of ATF6-N respectively (step 4). ATF6-N, traffics to the nucleus to transactivate genes required to restore ER homeostasis. The fate of the lumenal domain is unknown but maybe secreted. In the absence of ERp18, ATF6α rapidly exits the ER and is cleaved by an unknown post-ER localised protease to produce a fragment that is inefficiently processed by either S1P or S2P.
Figure 2(A) RPE1 cells were transfected with siRNAs as indicated, followed by serum starvation for 24 h, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm. (B) Quantification of the percentage of ciliated cells shown in (A). Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test. (C) Cells were transfected with siRNAs as indicated and incubated with HPβCD in serum-starvation media followed by filipin staining. Scale bar, 20 μm. (D) Cells were transfected with siRNAs shown in (C) and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm. (E) Quantification of the percentage of ciliated cells shown in (D). Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test. (F) Cells were transfected with siRNAs as indicated, incubated with cholesterol-MβCD complex in serum-starvation media, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm. (G) Quantification of the percentage of ciliated cells shown in (F). Data represent mean ± SD (n=3experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.
Figure 5Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with wild type B cells with or without 1μg α-GalCer and with or without agnostic anti-CD40. IgM was measured by ELISA of culture supernatants 4 days later. Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with anti-CD40 activated wild type B cells with or without 1μg α-GalCer and with blocking antibody to CD1d (1μg/mL, clone 1B1) or isotype control as indicated. IgM, IgG1, and IgA were measured by ELISA of 4 day culture supernatants. Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. IL-4 were measured by ELISA of culture supernatants. Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1 (D-E) were measured by ELISA of culture supernatants. Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1+ and IgA+ class switched B cells were enumerated by flow cytometry after 4 days of coculture Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1+ class switched B cells were enumerated by flow cytometry after 4 days of coculture
Figure 3A. Expression of endothelial junction protein ZO-1 and stabilization of ßcatenin in bEnd.3 cells treated with 30 nM F4L5.13 or 30 nM recombinant NDP for the indicated times. Blots are representative of five different experiments. Histogram represents the ratio of ZO-1 and ßcatenin levels over ß-Tubulin measured by densitometry of five independent experiments. Data are presented as mean ± SEM. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (*p<0.05 as compared with NT). B. Immunofluorescence of ZO-1, CLDN3 and CLDN5 localization at bEnd.3 cell junctions. bEnd.3 cells were treated or not with 30 nM F4L5.13 or 30 nM Norrin (NDP) with or without 100 ng/ml VEGF for 1h. ZO-1 is shown in green, CLDN3/5 in red and DAPI in blue. Scale bars: 15 µm. C. Quantification of ZO-1, CLDN3 and CLDN5 fluorescence intensity. Each column represents 40 measurements of fluorescence intensity per condition (y-axis). Data are presented as mean ± SEM. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (*p<0.05 as compared to VEGF treatment). D D. Transendothelial permeability assay quantifying the passage of FITC-dextran through a monolayer of bEnd.3 cells. Passage of FITC-dextran was measured after exposure of bEnd.3 cells to 100 ng/ml VEGF, 30 nM F4L5.13 or both or pretreated with VEGF for 1h before treating with F4L5.13 for 1h. Data are presented as mean ± SD, n=5 independent experiments. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (*p<0.05 as compared to VEGF treatment).
Fig.Figure 1(A) Phylogenetic tree of canonical human NHE1-9 (SLC9A1-9) cluster into those that localise predominantly to either the plasma membrane (pink) or endomembrane (orange); for completeness more distantly-related non-canonical human NHE members SLC9B1-2 (NHA1-2) (yellow) and SLC9C (blue) are also shown as labelled. (B) SEC trace and SDS-PAGE of purified NHE9* (residues 8 to and 574 out of 644) as depicted by schematic. (C) Activity of NHE9* co-reconstituted with ATPase into liposomes to mimic the in vivo situation (schematic). Representative ACMA fluorescence traces of liposome reconstituted NHE9* (blue), NHE9* double-mutant N243A-D244A (red), and rat fructose transporter GLUT5 (black). ATP-driven H+ pumping establishes a ΔpH (0-3 min). H+ efflux is initiated by the addition of 40 mM NaCl, and subsequent addition of NH4Cl (4 min) collapses the proton gradient. (D) Michaelis-Menten kinetics for NHE9* (red), NHE9 ΔCTD (green), N243A-D244A (blue) and rat GLUT5 (black) as detected by ACMA dequenching following substrate addition. In all experiments errors bars, s.e.m.; n = 3 technical repeats. The apparent KM values are an average from n = 3 separate protein reconstitutions. (E) Cryo-EM density map of the NHE9 ∆CTD homodimer with the 6-TM core transport domains (coloured in blue), the dimer domain (coloured in orange), and the linker helix (coloured in grey).
Model of Cvt and autophagy transport to the vacuole.Figure 1Model of Cvt and autophagy transport to the vacuole. There are many shared mechanistic features for the two pathways. 1, Cvt complex formation: prAPI is synthesized in the cytosol, oligomerizes into a dodecamer, and assembles into a Cvt complex; 2, double-membrane vesicle formation: the Cvt complex is enwrapped by a membrane source which, upon expansion and completion, forms the double-membraned Cvt vesicle (Cvt pathway) or autophagosome (autophagy); 3, vesicle targeting to the vacuole: the double-membrane vesicle is then targeted to the vacuole followed by fusion with the vacuolar membrane and release of the inner vesicle, termed the Cvt body (Cvt pathway) or the autophagic body (autophagy), into the vacuolar lumen. Subsequent breakdown of the Cvt or autophagic body releases prAPI into the lumen where it is proteolytically processed, in a proteinase A-dependent manner, to mature API. Major differences between the pathways include the following: 1, cargo selectivity: the Cvt pathway is a selective process and the Cvt vesicles appear to exclude bulk cytosolic components as cargo. In contrast, autophagy is nonselective and autophagosomes carry bulk cytosol, in addition to the Cvt complex, to the vacuole. prAPI is transported via both vesicle types. A recombinant, cytosolic marker protein, Pho8Δ60p, is transported to the vacuole via autophagy. Delivery of Pho8Δ60p to the vacuole can be monitored through cleavage of the COOH-terminal propeptide. 2, Vesicle size: Cvt vesicles are ∼150 nm in diameter, whereas autophagosomes are 300-900 nm in diameter. 3, Conditions for induction: the Cvt pathway is active in vegetative growth conditions while autophagy is induced under starvation conditions.
Effects of vernakalant on APs of hESC-ventricular and hESC-atrial CMs.Figure 7A Representative APs at 1 Hz of VM and AM CMs in response to vernakalant. Inset shows dV/dtmax.B, C Average APAplat (B) and dV/dtmax (C) in the absence and presence of vernakalant at 1-4 Hz. AP parameters are shown in Supplementary Table S6. Abbreviations as in Figs3 and 6.Data information: Data are presented as mean ± SEM. *P < 0.05 by Mann-Whitney rank-sum test for (B). P = 0.01, 0.008, 0.008 and 0.007, respectively, for comparison of APAplat between VM and AM groups at frequencies of 1.0, 2.0, 3.0 and 4.0 Hz. Two-way repeated measures ANOVA followed by pairwise comparison using the Student-Newman-Keuls test for (C). P = 0.686 between VM and AM groups and hence not statistically significant. For VM, P = 0.06 for 1 versus 2 Hz; P < 0.001 for 1 versus 3 Hz; P < 0.001 for 1 versus 4 Hz; P = 0.001 for 2 versus 3 Hz; P = 0.002 for 2 versus 4 Hz; and P = 0.857 for 3 versus 4 Hz. For AM, P = 0.03 for 1 versus 2 Hz; P < 0.001 for 1 versus 3 Hz; P < 0.001 for 1 versus 4 Hz; P = 0.02 for 2 versus 3 Hz; P = 0.02 for 2 versus 4 Hz; and P = 0.621 for 3 versus 4 Hz.
Figure 2A. 3T3-L1preadipocytes were adipogenically induced and lysates were prepared from cells collected at indicated days. Endogenous WDTC1 interaction with CRL4 subunits was evaluated by immunoprecipitation with anti-WDTC1 followed by immunoblot analyses with indicated antibodies. Fatty acid synthase (FAS) levels were monitored to evaluate induction. B-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Adipogenesis was assessed by Oil Red O staining (ORO) (B); plate (top) and microscopic (bottom) views, 0.1 mm scale bar. B-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Triglyceride levels were quantified by an enzymatic assay (C). B-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Adipogenic gene expression was evaluated by RT-qPCR analysis (D).