EMBO/sd-character-level-ner huggingface dataset:
Figure 2Analysis of the interaction between CtUba4 and GST-CtUrm1 (ratio 2:1 or 0.5:1 when marked with an asterisk) in the presence of nucleotide derivates by GST pull-down resolved by SDS-PAGE and visualized with Coomassie stain. Close up (for reference see full structure on upper left) of the CtUba4 linker (aa 286-321) highlighting important residues and the coordination of the zinc site (inset, lower left). Tabular summary of ATP hydrolysis by CtUba4 WT and mutants in the presence of CtUrm1. The amount of released PPi was measured using a fluorometric pyrophosphate assay kit. After PPi hydrolysis, Pi was also detected by a Malachite Green kit. Changes in enzymatic activity are indicated as a color gradient from blue to red, normalized to CtUba4 WT. PPi: pyrophosphate; Pi: inorganic phosphate; SD: standard deviation; n≥3 Functional analyses of ScUba4 mutant yeast using APM-gel retardation (top) and viability in response to rapamycin (bottom). PAGE was supplemented with APM to retard the migration of thiolated tRNAs and allow their visualization by northern blot using an anti-tRNA<sub>UU<em>C</em></sub><sup>Gl<em>u</em></sup>; probe. APM: ([N-Acryloyl-amino]phenyl) mercuric chloride; YPD: yeast extract peptone dextrose. All residue numbering follows the CtUba4 sequence, but the respective ScUba4 numbering is added in subscript.
Figure 1(A) Profile of transcript levels of autophagy-related genes in iBAT which are significantly modified in cold exposed mice (4ºC, 24h), compared with mice maintained at thermoneutral temperature (29ºC). (Fisher's exact test corrected by the Benjamini-Hochberg method, p< 0.05). Heatmap represents log-fold change of RPKM values of cold versus thermoneutral (TN) sample data obtained from RNAseq analysis. Each square represents replicates (n=4). The most repressed genes are shown on the bottom of the heatmap. Parkin expression in iBAT from mice exposed to cold (4ºC) for 1 day (1d) or 21 days (21d) compared with control mice (ctl) maintained at 22ºC. Left: Relative transcript levels. Right: representative immunoblot and Parkin protein quantification. Ponceau staining (PS) was used as loading control. (n=6; transcript levels) (n=3; protein levels). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, cold vs. control and ##p<0.01, 1d vs. 21d. One-way ANOVA with Tukey's post hoc test. Relative transcript levels of Park2 in sWAT and eWAT. (n= 6). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, cold vs. control and ###p<0.001, 1d vs. 21d. One-way ANOVA with Tukey's post hoc test. Relative transcript levels of Parkin in iBAT, sWAT and eWAT of mice injected with the β3-AR agonist CL316,243, every day for 1 week. (n=6). Data are presented as means ±s.e.m. *p<0.05, ***p<0.001, CL316,243 treatment vs. saline. Two-tailed unpaired Student's t-test.
Orm2 is retro-translocated by the Dsc complex and Cdc48 for proteasomal degradation.Figure 5(A) WT (pdr5∆) cells expressing FLAG-Orm2 and Sec61-GFP (integral ER membrane protein) treated with MG-132 were fractionated into insoluble membrane (P100) and soluble cytoplasmic (S100) fractions and analyzed by SDS-PAGE and Western blot with the indicated antibodies. WT cells, tul1∆ mutants (all in a pdr5∆ strain background) were left untreated or treated with the proteasome inhibitor (MG-132) as indicated and subsequently subjected to subcellular fractionation S100 and P100 fractions were subjected to denaturing FLAG-Orm2 immunoprecipitations (IP). Input and eluted fractions (with FLAG peptide) were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. WT cells and cdc48-3 mutants (all in a pdr5∆ strain background) were left untreated or treated with the proteasome inhibitor (MG-132) as indicated and subsequently subjected to subcellular fractionation S100 and P100 fractions were subjected to denaturing FLAG-Orm2 immunoprecipitations (IP). Input and eluted fractions (with FLAG peptide) were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. (D) P100 fractions from MG-132-treated WT (pdr5∆) cells expressing FLAG-Orm2 and Sec61-GFP were extracted with Na2CO3 solution at the indicated pH and subsequently fractionated by 100.000 x g centrifugation into soluble (S) and insoluble membrane (M) fractions, subjected to denaturing FLAG-immunoprecipitations and analyzed by Western blotting with the indicated antibodies. The asterisk indicates reactivity of the secondary antibody with IgG heavy chains. (E) Schematic representation of the degradation of Orm2 by the EGAD pathway.
Figure 3A) VPS15-ZZ was co-expressed with Ulk1 and Ulk2 wild type (WT) or kinase inactive (KI) before analysis by Western blot. An electrophoretic mobility shift in VPS15-ZZ was noted with WT only. B) VPS15 is depicted schematically, with the N-terminal myristate and pseudokinase, HEAT and WD40 domains. The 6 potential ULK substrates are annotated below in red. C) Alignment of human VPS15 sequences spanning each of the putative ULK substrates. Simplified rendering of the ULK consensus motif stipulating hydrophobics at positions -3, +1 and +2 is shown above (Φ = hydrophobic). These positions are highlighted in the sequence alignment (red -3, blue +1/+2) with the phosphoacceptor in green. D) Human VPS34 complex II homology model showing VPS15 (green), VPS34 (grey), Beclin1 (beige) and UVRAG (pink) and potential ULK substrates (red spheres). (Left) Y-shaped VPS34 complex II in standard orientation with N and C termini of VPS15 labelled. The blue dashed line represents a lipid membrane, positioned close to the membrane binding (left- and right-hand tips of Y) and lipid kinase (right hand tip of Y) domains of the complex. (Right) Rotated 90 degrees on horizontal axis towards point of view.
Figure 3Figure 3. CD22 is highly clustered on the surface of resting B cells(A-C) dSTORM analysis of CD22, CD19, IgM and IgD. Wild-type primary B cells were stained with Alexa647-conjugated antibody against CD22, Cy5-conjugated antibody against CD19 or IgD, or Cy5-conjugated Fab fragments against IgM and settled onto nonstimulatory coverslips. Cells were then fixed, imaged with dSTORM and analysed.(A) 2D (top and middle) and pseudo 3D (bottom) dSTORM images were reconstructed from single molecule localizations. The white dashed square is shown in magnification (middle) and pseudo 3D (bottom).(B and C) Quantification of the distribution of CD22, CD19, IgM and IgD with H function and Hopkins index. Error bars (Hopkins index) and thin lines (H function) denote mean ± SEM. Data are pooled from at least three experiments per receptor.*P < 0.001, **P < 0.0001 (Student's t-test).
Rhabdoid tumor is sensitive to mithramycin, but not general chemotherapeutic agents.Figure 1A: Graph of the ratio of the median IC50 of the entire panel to that of each cell line generated from a screen of 23 pediatric cancer cell lines. Rhabdoid tumor cell lines (red) cluster towards the left of the graph indicating these cell lines are more sensitive to mithramycin. These results confirm a previously published screen (Osgood et al., 2016). B: Graph of the ratio of the median IC50 of the entire panel to that of each cell line generated from a published screen of 445 agents in 62 sarcoma cell lines (Teicher et al., 2015). The rhabdoid tumor cell line, G401 (red), appears on the left side of the graph indicating this cell line is more sensitive to mithramycin. C: Dose response curves of rhabdoid tumor and Ewing sarcoma cell lines. RT cell lines (black) are sensitive to mithramycin treatment with a similar IC50 value as TC32 ES cells (grey). RT cell lines are not sensitive to three broadly-active chemotherapeutic agents: etoposide, doxorubicin or SN38. Data represents mean with standard deviation derived from three independent experiments.
FRAP analysis reveals that the diffusion of UIS4‐mCherry is restricted in most LS‐TVN extended membrane clusters, but free within the PVM.Figure 6A and B) The bleach was applied to elongated membrane clusters of the LS‐TVN approximately 27 h post‐infection. The UIS4‐mCherry signal typically did not recover in the bleached area (A), although it was found to recover in some elongated membrane clusters (B). C) When the bleach was applied to the PVM at 25 h post‐infection, the signal recovered rapidly in the bleached region. Upon the application of additional bleach pulses in the same area, the bleached area (region #1 measured graphically) repeatedly recovered, and the remaining PVM‐associated UIS4‐mCherry signal depleted (region #2) although the LS‐TVN‐associated signal remained (region #3). When a region of the LS‐TVN was subsequently bleached (yellow arrow, region #4), no recovery was observed. The times are shown in minutes and seconds. Scale bars, 10 µm.
Figure 4A. Domain organization of Sec63. The sequence of the acidic patch on the C-terminus - divided into C1 and C2 regions - is shown in the lower panel. The two phosphorylated threonines (T652 and T654) are marked in the C2 region. The J domain of Sec63 is indicated by "J". B. Pull-down assay of Sec62 domain with different biotin-labeled Sec63 C-terminal peptides. Sec63-C2P denotes di-phosphorylated Sec63-C2. C. ITC-thermogram obtained from ITC after titration of Sec62 domain with increasing amounts of Sec63-C2P. D. Difference in HDX between the Sec62 domain in complex with Sec63-C2P and the Sec62 domain alone. The graph depicts the summed means of differences ± 3 SD (n = 3 technical replicates) for each peptide plotted according to the midpoint of each peptide. E. Crystal structure of Sec62 domain color coded according the difference in HDX The critical residues constituting the interaction interface between Sec63-C2P and Sec62 are labelled. F. Pull-down assay of the Sec62 domain and a mutant in the interaction interface (Sec62R51E) with biotin-labeled Sec63-C2P.
/K29-linked ubiquitination of MAVS (A) Immunoprecipitation analysis in HEK293T cells expressing Flag-MAVS and Myc-Ub together with HA-RNF34 or HA-H342A.Figure 3(A) Immunoprecipitation analysis in HEK293T cells expressing Flag-MAVS and Myc-Ub together with HA-RNF34 or HA-H342A. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. The levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results. (B) Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS and HA-RNF34 together with Myc-Ub (K6, K11, K27, K29, K33, K48, or K63 only) as indicated. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results. (C) Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS and HA-RNF34 together with Myc-Ub (wild-type, WT; Lys-to-Arg mutants of all Ub lysines, KO; K27/29R). Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results. (D) Immunoprecipitation analysis of K27 ubiquitination of MAVS in HEK293T cells infected with VSV for the indicated times. Anti-MAVS immunoprecipitates were analyzed using immunoblotting with an anti-MAVS or anti-Ub-K27 antibody. The WCL was immunoblotted with an anti-MAVS or anti-Ub-K27 antibody. Cell-based studies were performed independently at least three times with comparable results. (E) Immunoprecipitation analysis of siCtrl- or siRNF34-1-transfected cells infected with VSV for the indicated times. Anti-MAVS immunoprecipitates were analyzed using immunoblotting with an anti-MAVS or anti-Ub-K27 antibody. The WCL was immunoblotted with an anti-MAVS or anti-Ub-K27 antibody. Cell-based studies were performed independently at least three times with comparable results.
PolyUb proteins are cleared from PML-NBs by chaperones (see also and Appendix -5).Figure 5PolyUb protein (FK1) labelling in PML-GFP HeLa Kyoto cells that were either left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Cells were either immediately fixed or let to recover for 5 h in drug-free medium (Control), with VER (40 µM) or Eeyarestatin I (5 µM). Scale bars: 10 µm. Quantitation of the % of cells with polyUb (FK1) nuclear foci shown in (A). Number of cells counted/condition: 310 - 1258 in three independent experiments; statistical significance via One-way ANOVA; p = 0.0003 or 10-6, ± s.e.m. Quantitation of the number of PML-NBs enriched for polyUb (>2); n = 611 - 2612; statistical significance via One-way ANOVA; p < 10-10. Automated PML-NB segmentation is based on PML signal. Quantitation of the % of cells with polyUb proteins (FK1) sequestered in nuclear foci. HeLa cells were treated with OP-puro (25 µM) and heat shock (HS) at 42˚C for 2 h. Then, cells were either immediately fixed or let to recover for 3 h in drug-free medium (control), with VER (40 µM) or Eeyarestatin I (5 µM). Number of cells counted/condition: 593 - 1016 in three independent experiments; statistical significance via One-way ANOVA; p = 10-3, ± s.e.m. Quantitation of the number of PML-NBs enriched for polyUb (>2) in HeLa cells treated as described in (D) but with a recovery time of 2 h; n = 1155 - 3283; statistical significance via One-way ANOVA; p < 10-10. Automated PML-NB segmentation is based on PML signal. Amylo-Glo staining of PML-GFP HeLa Kyoto cells that were subjected to the following treatments: untreated (Control); MG132 (10 µM) and OP-puro (25 µM) for 4 h, alone or combined with CHX (50 μg/ml). Where indicated cells were allowed to recover for 5 hrs in drug-free medium (Control) or with VER (40 µM). Confocal images showing Amylo-Glo and PML-GFP. Scale bars: 5 µm. Amylo-Glo staining of PML-GFP HeLa Kyoto cells that were subjected to heat shock (HS) at 42˚C for 2 hrs. Confocal images showing Amylo-Glo and PML-GFP. Scale bars: 5 µm.
Figure 1A. The Armi complex immunoisolated from OSCs using anti-Armi antibodies contains Yb, SoYb, and Vret. B. The Vret complex immunoisolated from OSCs using anti-Vret antibodies contains Yb, Armi, and SoYb. C. Silver staining of protein components in the Vret complex The lane α-Vret (left) contained only the anti-Vret antibodies used in these experiments. D. Immunoprecipitation and western blotting were performed in OSCs upon RNAi treatment (kd). Even without the association of Yb, Armi, SoYb, and Vret associated. Yb and Armi remained bound to each other upon SoYb or Vret depletion. The level of SoYb (input) decreased upon Vret depletion. Likewise, the level of Vret (input) decreased upon SoYb depletion. E. Upon Armi depletion, SoYb and Vret were still bound to each other but this heterodimer no longer associated with Yb. F. Immunofluorescence analyses in OSCs. Yb bodies disappeared upon Yb depletion, which led Armi, SoYb and Vret to be localized in the cytosol. Yb bodies were detected in OSCs upon Armi depletion, but SoYb and Vret localized in the cytosol. Armi was detected at Yb bodies upon SoYb or Vret depletion. Yb bodies are indicated by white arrows. Nuclei are shown in blue. Scale bar: 2 μm. G. The hierarchy of Yb body assembly proposed in this study. Domain structures of Yb, Armi, SoYb, and Vret are shown on the right. Hel-C, Helicase C-terminal; eTud, extended Tudor; ZnF, zinc finger; RRM, RNA-recognition motif; MYND, MYND-type zinc finger.
Correlative light and electron microscopy shows distinct features of type A and C cellular neoaggregates Light microscopy images (LM) showing the HA-immunoreactive inclusions (green color, black arrowheads) identified in ultrathin sections from resin-embedded cell monolayers.Figure 5Light microscopy images (LM) showing the HA-immunoreactive inclusions (green color, black arrowheads) identified in ultrathin sections from resin-embedded cell monolayers. The LM images were overlaid with low magnification EM images (EM/LM overlay) to localize the same region within the cells for EM imaging at higher magnification (dotted black square, EM). A) HA-immunopositive regions within cells seeded with FTLD-TDPA patient material showed accumulated filaments (examples indicated with white arrowheads). B) HA-immunopositive regions within cells seeded with FTLD-TDP-C patient material showed aggregated amorphous material. Scale bars LM and EM/LM overlay, 10 µm; EM, 500 nm
Figure 7(A, C) WT BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times. Cell lysates were immunoprecipitated with anti-MK2, and levels of PML were determined. Mk2-/- BMDMs served as a negative control. (B) WT BMDMs were treated with TNF (100 ng/ml) for the indicated times, before immunoprecipitating p38 MAPK and determining the levels of PML, p38, MK2 in the precipitates and total cell lysates. (C) WT BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times. Cell lysates were immunoprecipitated with anti-PML, and levels of p38 and MKK3 were determined. Pml-/- BMDMs served as a negative control. (D) WT and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times. p38 MAPK was immunoprecipitated, and its association with MK2 was then analysed. (E) Recombinant Flag-PML-I, GST-MK2 (60 ng) and His-p38 (60 ng) were incubated, as indicated, at 4 °C for 2 h. p38 was pulled down by anti-p38 and then PML, MK2 and p38 contents in the precipitate and reaction mixture (input) were detected by immunoblotting. (F) Recombinant Flag-PML-I, GST-MK2 (100 ng) and His-p38 (200 ng) were incubated, as indicated, at 30 °C in in vitro kinase assay buffer (20 mM HEPES, 10 mM MgCl2, 100 nM ATP, 1 mM DTT) for 20 min. The levels of p-MK2, MK2, p38, and PML (FLAG) in the reaction mixture were then determined.
Figure 3(A) Surface calreticulin on MM cells was evaluated by flow cytometry after exposure to 800 nM BoxA or 1 μM MTX for 4 or 6 hours. Statistics: One-way Anova with Dunnett's post-test; n=3. Data information: columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (B) Representative immunohistochemical staining for calreticulin in tumor masses of control (PBS) and BoxA-treated mice. Scale bar 50 μm. The distribution of calreticulin positive pixels describes the location of calreticulin (intracellular vs ecto-calreticulin). Statistics: Mann-Whitney test, n=4. (C) HMGB1 levels in the medium of AB1 cells treated with BoxA or 1 μM MTX for 24 hours. Statistics: One-way Anova with Dunnett's post-test; n=3. Data information: columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (D) Ratio of phosphorylated eIF2α (p-eIF2α) over eIF2α in MM cells treated with increasing concentrations of BoxA or 1 μM MTX for 24 hours; β-actin is shown as loading control. Statistics: One-way Anova with Dunnett's post-test; n=2. Data information: columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (E) MM cells (3x105) were grown for 16 hours before adding increasing concentrations of BoxA or 1 μM MTX, and then further grown for 24 hours; t-16h indicates the number of cells at the start of the experiment. Data information: columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (F) Western blot analysis of cleaved caspase-3 in MM cells exposed or not to BoxA or MTX for 24 hours; α-tubulin was used for normalization. Statistics: One-way Anova with Dunnett's post-test; n=2. Data information: columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (G) Confocal immunofluorescence microscopy of cleaved caspase 3 (red) in MM cells treated for 24 hours with MTX (1 μM) or increasing concentrations of BoxA. Nuclei were stained with Hoechst (blue) and cytosol with phalloidin (green). Scale bar 50 μm. (H) Tumor growth detected via BLI. MM cells pretreated or not with 800 nM BoxA overnight were injected in mice (PBS group, n=7; BoxA pretreated, n=6). (I) Kaplan-Meier survival curve of mice injected with MM cells pretreated with PBS or BoxA shown in panel H (Log rank Gehan Breslow Wilcoxon test, p=0.59).
a) A schematic diagram depicting incompatibility of idealized cone-shaped lipids with planar assemblies.figf2(b) Filling the defects with inverted cone-shaped lipids inhibits lipidation. In vitro coupling reactions of GL1 were run on sonicated liposomes containing 30 mol% DOPE (2 mM total lipid) as in Fig. 1. To determine whether inverted cones influenced the reaction, increasing amounts of stearoyl-CoA were added immediately before initiating the reaction with ATP. Quantification is of n = 3 samples; error bars represent standard deviation. P values represent a comparison with samples without stearoyl-CoA. **P 0.1; *P 0.05. (c) Altering the shape of PE to reduce local membrane defects inhibits the lipidation reaction. In vitro coupling reactions of GL1 were run on liposomes of three different sizes (400 nm and 50 nm extruded liposomes and sonicated liposomes) and of three different lipid compositions. Lipid compositions include 30 mol% DPPE, DSPE or DOPE (see Methods for full definitions), 10% bl-PI, and POPC. Total lipid = 2 mM. Quantification is of n = 3 samples; error bars represent standard deviation. The extent of lipidation in b and c is plotted as a percentage of total GL1 as determined by densitometry. P values represent comparison with most active liposomes (sonicated DOPE). **P 0.1; *P 0.05.
Figure 4Spinal cord (C8-level) of MIRAS patient, neurofilament staining. Dorsal columns, especially the gracile, show pallor (star); motoneurons in the anterior horns are preserved (black arrow). Scale bar 1 mm. Oculomotor complex of POLG patient with progressive ophthalmoplegia; midbrain level. Hematoxylin-eosin staining. The whole area shows spongiotic degeneration. Scale bar 50 µm.
Figure 5In order to validate the modeling and test its predictive power, we repeated the competition experiments between Aci2 and Pa across a 54-condition grid of resource supply ratios and dilution factors Shown is the fraction Pa on Days 1, 3, 5, and 7 for eight total replicates of each condition (two biological replicates, each with four technical replicates started at different initial species fractions We used the model to predict steady-state population fractions for each condition. We calculated mean final population fractions from the experimental data and compared these fractions to the model predictions.