EMBO/sd-character-level-ner huggingface dataset:
Figure 3MITOL KO facilitated IRE1α oligomerization. MEFs were transfected with IRE1α-GFP expression vector for 24 hours prior to Tu treatment for indicated periods (A). The right panels show 5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection in each independent experiment (A). To evaluate the oligomerization level of endogenous IRE1α, cells without any transfection were solubilized and separated by sucrose density-gradient centrifugation, followed by immunoblotting with anti-IRE1α antibody (B). IRE1α oligomers were stabilized by MITOL KO. MEFs transfected with IRE1α-GFP were treated with Tu. After 4 hours, these cells were washed with PBS and subsequently re-fed with fresh media for indicated periods. The right panels show 5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection at indicated periods after wash out of Tu in each independent experiment. Scale bar represents 10 µm. Sustained IRE1α autophosphorylation after wash out of Tu in MITOL-KO MEFs. MEFs were treated with Tu for 4 hours and washed with PBS and re-fed with fresh media for indicated periods, following by immunoblotting anti-IRE1α antibody after Phos-tag SDS-PAGE. Error bars represent SD (n=3). MITOL deletion promoted the interaction between IRE1α and BIM under ER stress. MITOL-depleted HEK293 cells (KO) were generated using CRISPR-Cas9 system. Cells were co-transfected with indicated vectors 24 hours prior to Tu treatment for 12 hours. Cells were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies.
Impact of decreasing procaspase-8 on DISC assembly and caspase activation.Figure 2(A) Western blot analysis of FADD, FLIP and Caspase 8 at the TRAIL-R2 DISC in HCT116 cells treated with escalating doses (0-30nM) of caspase-8 siRNA for 48 hours. Samples were incubated with anti-TRAIL-R2 (AMG655) beads (4x) for 90 minutes prior to collection. A scrambled control siRNA (SC) was transfected (30nM) for comparison. (B) Quantification of Caspase 8 (p55, p43/41 and p26/24) and FLIP (FLIP(L), FLIP(S) and p43-FLIP) at the TRAIL-R2 DISC by densitometry and normalised to known protein standards (C) Ratio of Caspase-8:FLIP calculated from values presented in (B). (D) Caspase-8 (IETDase) and -3/7 (DEVDase) activity assay in the unbound soluble fraction (E) Western blot analysis of FLIP, Caspase 8, and FADD recruitment to the TRAIL-R2 DISC in PC3 cells stably expressing an empty vector (EV) or flag tagged FLIP L (FL) treated with 2.5µM MS-275 for 48 hours, followed by a 90 minute DISC IP. * modified form of FLIP, potentially mono-ubiquitinated p43-FLIP(L). (F) Table of the relative ratios of Caspase-8 (p55, p41/43 and p24/26) and FLIP (endogenous and Flag-tagged) at the TRAIL-R2 DISC in MS-275 treated and untreated cells after a 90 minute DISC IP. (G) Caspase-8 (IETDase) activity assays of the unbound soluble fraction
Figure 1A. MITOL interacts with Parkin under CCCP treatment. HeLa cells or HeLa cells stably expressing HA-Parkin were transfected with the indicated vectors and treated with DMSO or CCCP (10 µM) for 4 h. Lysates of cells were subjected to an IP assay with anti-myc antibody and IB assay with the indicated antibodies. B. Interaction of endogenous MITOL and Parkin in HeLa cells stably expressing HA-Parkin. HeLa cells stably expressing HA-Parkin were treated with DMSO or CCCP (10 µM) for 12 h and subjected to an IP-IB assay with the indicated antibodies. C. PINK1 knockdown decreases interaction between MITOL and Parkin. HeLa cells stably expressing HA-Parkin were transfected with PINK1 siRNA or control siRNA and treated with DMSO or CCCP (10 µM) for 4 h. MG132 (30 μM) was added 3 h after each treatment. Lysates of cells were subjected to an IP assay with anti-myc antibody and IB assay with the indicated antibodies. D. MITOL overexpression enhances the ubiquitination of Parkin. HeLa cells or HeLa cells stably expressing HA-Parkin were transfected with the indicated vectors and treated with DMSO or CCCP (10 µM) for 8 h. MG132 (30 μM) was added 3 h after each treatment. Lysates of cells were subjected to an IP-IB assay with the indicated antibodies. WT, wild-type; CS, MITOL C65/67S mutant lacking ubiquitin ligase activity. E. MITOL fails to ubiquitinate the Parkin K220R mutant. HeLa cells were transfected with the indicated vectors and treated with CCCP (10 µM) for 8 h. MG132 (30 μM) was added 3 h after CCCP treatment. Lysates of cells were subjected to an IP-IB assay with the indicated antibodies and compared ubiquitination levels of WT, K129R, K220R, and K427R HA-Parkin. F. Parkin is ubiquitinated by K48-polyubiquitin in mitophagy. HeLa cells stably expressing HA-Parkin were treated with CCCP (10 µM) for 48 h together with MG132 (30 µM) for the final 45 h. Parkin was immunoprecipitated from cell lysates and then performed ubiquitin restriction assay with AMSH, OTUB1, and USP2 using UbiCRest kit. Lysates of cells were subjected to an IB assay with the indicated antibodies. DUBs, deubiquitinating enzyme. G. Endogenous MITOL enhances the K48-polyubiquitin chains of Parkin. WT or MITOL KO HeLa cells stably expressing HA-Parkin were treated with CCCP (10 µM) for indicated time. MG132 (30 μM) was added 3 h after CCCP treatment. Lysates of cells were fractionated into cytosolic, mitochondrial and ER fractions, and then subjected to an IP-IB assay with the indicated antibodies. K48-polyubiquitin chains were immunoblotted with K48-specific ubiquitin antibody.
Figure 2WT, Abro1−/− and Nlrp3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β and TNF-α in the peritoneal lavage fluid (A). n = 6 for PBS groups and n = 12 for MSU groups. WT, Abro1−/− and Nlrp3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. Flow cytometric analysis of peritoneal cell exudates (B). n = 6 for PBS groups and n = 12 for MSU groups. Abro1+/+ and Abro1−/− mice were intraperitoneally injected with MSU (1 mg per mouse). Flow cytometric analysis of caspase-1 activity in peritoneal exudate monocytes and neutrophils. n=12 per group. Abro1+/+ and Abro1−/− mice were intraperitoneally injected with Alum (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β in the peritoneal lavage fluid (D) n = 6 for PBS groups and n = 12 for Alum groups. Abro1+/+ and Abro1−/− mice were intraperitoneally injected with Alum (1 mg per mouse) or vehicle (PBS) for 6 h. flow cytometric analysis of peritoneal cell exudates (E). n = 6 for PBS groups and n = 12 for Alum groups. Survival of Abro1+/+ and Abro1−/− mice subjected to LPS (15 mg/kg). n=20 per group. ELISA of serum IL-1β from Abro1+/+ and Abro1−/− mice 3 h after intraperitoneal injection of LPS (15mg/kg) or vehicle (PBS). n = 6 for PBS groups and n = 12 for LPS groups.
Binding capacity of the RBD of S proteins of four SARSr-CoV viruses to ACE2 of potential hosts.Fig 4.Free energy (kcal/mol) for the binding between S proteins of four SARSr-CoV viruses and ACE2 of four likely hosts. Molecular interactions of RBD of the S proteins of 2019-nCoV and Pangolin-CoV with ACE2 proteins of humans and pangolins. The key residues participating in the hydrogen bond formation at the interface between ACE2 and S protein are indicated by red arrows.
Fig.Figure 5(A) Structural alignment of the yeast Mrp17 (from PDB:5MRC) and its E. coli homolog ribosomal protein S6 (from PDB: 6WD0) viewed from its cytosol-facing side, highlighting regions of structural homology (blue and yellow) and non-homologous C-terminal regions (grey), Tom20-binding domains in Mrp17 are highlighted in green, schematic summaries of such structural alignments for all MRPs and their bacterial homologs are shown in Appendix Fig. S7. (B) Average MTS prediction scores (MitoFates and TargetP2) for structurally conserved yeast MRPs and their bacterial homologs sorted by the difference between bacteria and mitochondria. (C) Expression of structurally conserved bacterial RPs fused to GFP in yeast cells (Full micrographs in al channels are shown on Fig. EV5). Scale bar is 10 µm. (D) Schematic summary of structural alignment of bacterial S6 and yeast Mrp17 shown in panel (A) highlighting internal targeting signal (grey outline), additional features of Mrp17 structure (two TBMs), and six glutamate residues (E's) at the C-terminus of bacterial S6 (top); schematic of chimeric constructs of EcS6: S6/Mrp17ITS with Mrp17 amino acids 30-60 incorporated instead of the homologous region of S6 and fused to DHFRmut (middle), S6/Mrp17ITS+ that is similar to S6/Mrp17ITS but has 7 amino acids mutated to lysines (bottom). (E) Chimeric variants of S6 depicted in panel D imported into isolated yeast mitochondria. Lanes are labeled as described in Fig. 2 legend. (F) Mrp17 targeting and translocation model that requires Tom20-binding motifs and positive charges for targeting (STEP 1) and relies on ubiquitous positive charges for translocation (STEP 2).
Figure 5Silver nitrate-stained NuPAGE gel of hnRNPLL interacting proteins. Red star indicate hnRNPLL specific interacting proteins compared to IgG. Mass spectrum identification of the hnRNPLL interactome in ES cells. RNA splicing associated proteins are well enriched by hnRNPLL IP. The enrichment score was log2-transformed before plotting. Western blot validation the interactions between hnRNPLL and the indicated splicing associated proteins. Cumulative distribution of PSI with exon skipping events between WT and hnRNPLL-KO ES cells. Kolmogorov-Smirnov test was used to determine the significance. Volcano plot of differential exon skipping events between WT and hnRNPLL-KO ES cells. Indicated AS events were labeled in purple (up-regulated) and green (down-regulated) with gene names. The regional distribution of differential exon skipping events between WT and hnRNPLL-KO ES cells. Western blot analysis of hnRNPLL protein during EB formation of WT ES cells at indicated days. Venn plot showed the divergent exon skipping events between EB differentiation and hnRNPLL deletion. 38 exon skipping events that were inhibited by hnRNPLL but increased during EB differentiation, and 25 events that showed the opposite profile. Heatmap of the 38 and 25 divergent exon skipping events in Figure 5I. * indicates the different exon skipping events of the same gene. RT-PCR validation of partial divergent exon skipping events shown in Figure 5J. Red boxes represent ESC preferred exons, blue boxes indicate EB preferred exons.
Overexpression of Ago1x increases global translation (A) Ribopuromycylation assay showing global translation profile of HeLa cells overexpressing FLAG-HA-Ago1 or FLAG-HA-Ago1x.Figure 8Ribopuromycylation assay showing global translation profile of HeLa cells overexpressing FLAG-HA-Ago1 or FLAG-HA-Ago1x. Densitometric analysis of four independent experiments is shown as bar graph (mean ± S.E.). Puromycin densities were normalized to ponceau S stain densities. *, P < 0.029 (Mann-Whitney test). [35S]-methionine autoradiogram showing global translation profile of HeLa cells overexpressing FLAG-HA-Ago1 or FLAG-HA-Ago1x. Densitometric analysis of three independent experiments is shown as bar graph (mean ± S.E.). Autoradiogram densities were normalized to Coomassie stain densities. *, P < 0.029 (Mann-Whitney test).
Nix recruits EGFP‐GABARAP‐L1 to stressed mitochondria in reconstituted Nix−/− MEFs.Figure 3Nix recruits EGFP‐GABARAP‐L1 to stressed mitochondria in reconstituted Nix−/− MEFs. Nix−/− MEFs cotransfected with EGFP‐GABARAP‐L1 and wt‐Nix (A), or Nix‐W35A (B), were incubated with and without mitochondrial poison CCCP for 3 h. Cells were then fixed and analysed for colocalization between EGFP‐GABARAP‐L1 and Nix (stained with Flag antibodies). The final panel in each row shows colocalization between Flag‐Nix‐Cy3 and GABARAP‐L1 using the 'colocalization' highlighter plug‐in for ImageJ software (NIH, Bethesda, MA, USA). (C) Quantification was achieved by counting the number of complete colocalizations, EGFP/Cy3 per cell for approximately 30 cells per condition. Results are expressed as a percentage of colocalization (EGFP:Cy3) in control and CCCP‐treated cells. Error bars in (C) show the standard deviation (s.d.) of three independent experiments. Statistical analysis was performed using the Mann-Whitney t‐test. CCCP, carbonyl cyanide m‐chlorophenyl hidrazone; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescent protein; MEF, mouse embryonic fibroblast; wt, wild type.
Figure 1A, Reconstructed MSOT images (800 nm) without and with linear unmixing data in the neck and the upper and lower abdominal areas, showing the ROIs for the liver, kidney, iBAT, rpWAT, aorta, and SV. Unmixing result: blue for Hb, red for HbO2, yellow for lipid. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (maximum value neck/upper abdominal/lower abdominal: Hb: 2.5/3.5/0.9; HbO2:2.9/4.9/2.5; Lipid: 24000/19000/13000) Scale bar: 4 mm. B, Normalized spectra of liver, kidney, iBAT, rpWAT, aorta, and SV. Data represent the mean (+/- 95% confidence) from 8 animals (n = 8). Data information: In the figure, A.U. = arbitrary units. C, Normalized spectra of liver, kidney, iBAT, and rpWAT in vivo and ex vivo. The two spectra in each plot are from the same animal. Data information: In the figure, A.U. = arbitrary units. D-E, Linear unmixing results from liver, kidney, iBAT, and rpWAT. Each dot represents data from one animal, in total 8 animals (n = 8). Data represent the mean (+/- 95% confidence). The unpaired t test was used to verify the statistical significance. sO2 liver vs kidney: P = 1.66E-07; TBV iBAT vs rpWAT: P = 5.58E-07 Data information: In the figure, A.U. = arbitrary units.
Figure 5(A) Schematic representation of the phosphosite (Ser439) identified in PIN2 in our study. PIN2 protein possesses three specific regions: a region with five transmembrane segments (residues 1-163), a central hydrophilic loop extending from residue 164 to 482 and a region with five additional transmembrane segments (residues 483-647). Protein alignment with PIN2 or PIN-like proteins from different species indicates conservation of the serine residue in eudicot, monocot, and gymnosperm species. The red arrow highlights the S439 in the central loop of PIN2 proteins in different plants. The size of each protein in amino acids and the percentage of identity against the Arabidopsis PIN2 are indicated. (B) Levels of PIN2 phosphopeptide in our experiments. Bars represent the mean plus standard error of replicates (2 biological replicates for nitrate treatments at 20 min (Col-0 roots) and 3 biological replicates for all other experimental conditions). Each independent biological replicate consisted of a pool of 4.500 roots collected from Arabidopsis plants grown independently under the same experimental conditions. The asterisk indicates statistically significant differences in phosphoproteomic analysis (multiple t-test comparison without testing corrections, assuming same variance, p < 0.05). (C) Detection of phosphorylation PIN2 by Phos-tag Western blotting. Arabidopsis plants (Col-0) were growth in ammonium as only nitrogen source, and treated with 5 mM KNO3 or 5mM KCl as control. Total protein from roots were analyzed in SDS PAGE using Phos-tag to detect changes in phosphorylation status. Immunoblotting was performed with PIN2 antibody. Total proteins isolated from eir1.1 roots were used as a negative control. White and red asterisks indicate a slow- or fast-mobility band corresponding to a more or less phosphorylated PIN2, respectively. (D) Western blot against PIN2 protein comparing nitrate treated (KNO3) and control (KCl) condition in Arabidopsis roots for all genotypes (eir1-1 mutant background was complemented with PIN2::PIN2wt-GFP (PIN2wt), PIN2::PIN2S439D-GFP (PIN2S439D phospho-mimic point mutation) or PIN2::PIN2S439A-GFP (PIN2S439A, phospho-null point mutation). (E) Time-course analysis of PIN2 mRNA levels in response to nitrate treatments in Arabidopsis roots. Bars represent the mean plus standard deviation of 3 biological replicates.
The reduced incorporation of α-synuclein inclusions into autophagosomes by autophagy inhibition.Figure 3(A) HEK293 cells stably expressing GFP-LC3 were transfected with control (upper) or Atg-5 siRNA (lower). After 36 h, α-synuclein fibrils were introduced into them for 4 h, followed by immunostaining with anti-phospho-α-synuclein antibody (P-αSyn). Blue, DAPI. Scale bar, 10 µm. (B) The number of GFP-LC3-positive α-synuclein inclusions (%) were assessed in randomly chosen fields (n = 5) with 48-141 inclusions. Statistical analysis was performed with t-test (*p<0.01). (C) Clearance of α-synuclein inclusions in control (solid bar) or Atg-5 (open bar) knockdown cells was measured at 4 h and 24 h after α-synuclein fibrils. The number of phosphorylated α-synuclein-positive cells was assessed in randomly chosen fields (n = 5). Data from each experiment are were normalized to Cont-siRNA-4 h (100%) and represented as relative value of α-synuclein inclusions ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01
Figure 4(A-B) The indicated cell lines were transfected with either miR-515-5p or a non-targeting miR (miR NC) together with a MARK4 expressing or an empty vector (EV) plasmid DNA. MARK4 over-expression rescues the inhibition by miR-515-5p of random (A) and directed (B) migration of the indicated cell lines. (A-left panel) Plots show overlays of representative trajectories described for MDA-MB-231 cells (n=30). (A-right panel) The migration distance was quantified and represented as the normalised mean ± SEM. (B-left panel) Representative field of view from the bottom side of the transwell membrane for MDA-MB-231. (B-right panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n=5 fields per condition). Data are mean of three experiments ± SEM. (A-C) P values were calculated by t-test between the transfected and the respective control condition (EV+miR-NC) (*,P<0.05; **,P<0.01; ***,P<0.001).
Figure 3PIP strip lipid binding assay with recombinant PTPIP51_ΔTM, PTPIP51_TPR, and PTPIP51_ΔTPR. PA binding of PTPIP51 (PTPIP51_ΔTM and PTPIP51_TPR) was monitored by a PA precipitation assay and a liposome flotation assay. The POPC:POPE:POPA ratios in each liposome were 50:50:0, 50:40:10, 50:30:20, and 50:20:30. Diagrams of the liposome precipitation and flotation assays are illustrated separately. The PA transfer activity of PTPIP51 (PTPIP51_ΔTM and PTPIP51_TPR) was monitored by a FRET-based lipid transfer assay. A schematic cartoon of the FRET-based in vitro lipid transfer assay is presented on the left. a.u.: arbitrary unit. The alternative bead pulldown-based lipid transfer assay with PA and MLCL. A schematic cartoon representation of the fluorescent lipid transfer assay is shown on the left. The y-axes in the graphs show the percentage of acceptor liposome fluorescence calculated using the following formula: 100×Facceptor/(Facceptor+Fdonor).
a) Bif-1+/+ and Bif-1−/− MEFs were cultured in EBSS for 0 or 6 h and subjected to immunoprecipitation with anti-Bif-1 monoclonal antibodies.figf5(a) Bif-1+/+ and Bif-1−/− MEFs were cultured in EBSS for 0 or 6 h and subjected to immunoprecipitation with anti-Bif-1 monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with the indicated antibodies. (b) 293T cells were cotransfected with the indicated plasmids for 24 h and subjected to immunoprecipitation with anti-Flag monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Myc or anti-Flag polyclonal antibodies. (c) 293T cells were cotransfected with the indicated plasmids for 22 h, starved in EBSS for 2 h and subjected to immunoprecipitation with anti-Flag monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Myc or anti-Flag polyclonal antibodies. (d) 293T cells were transfected with GFP siRNA or UVRAG siRNA for 24 h, starved in 2 h for 2 h and subjected to immunoprecipitation with anti-Bif-1 monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Bif-1 and Beclin 1 polyclonal antibodies. The reduction of UVRAG protein expression by UVRAG siRNA was confirmed by immunoprecipitation and immunoblot analysis with anti-UVRAG polyclonal antibody. Uncropped images of the immunoblots are shown in the Supplementary Information, Fig. S5.
Model of single β-catenin mutation and interaction with E-cadherin in the small intestine and colon.Figure 7Model of single β-catenin mutation and interaction with E-cadherin in the small intestine and colonA, B Model of a single activating β-catenin mutation in the murine small intestine and the colon. In contrast to the small intestine (A), the increased levels of E-cadherin in the colon complexed with mutant β-catenin prevent its accumulation in the nucleus (B).
Figure 1A Strategy for identifying metabolic plasma biomarkers and, subsequently, therapeutic opportunities for LAM based on the analysis of lung-metastatic breast cancer gene expression data B Significantly enriched metabolic pathways among the 30 enzymes identified in the previous analysis of TSC2 expression correlations. C Overabundance of MIAA in LAM plasma. Top panel shows each control and patient group (number (n) of samples are indicated); bottom panel shows aggregation of related pulmonary diseases. The asterisks indicate significant differences based on two-sided Mann-Whitney tests (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; top panel, control-LAM P = 0.048, LAM-Langerhans P = 1x10-4, LAM-Sjögren P = 1x10-4, LAM-lupus P = 1x10-3, LAM-emphysema P = 0.041; bottom panel, LAM-Langerhans P = 7x10-6 and control-other P = 0.031). Average values are indicated with lilac-colored lines. Comparison of the three groups: Kruskal-Wallis test P = 6x10-4 (top panel) and P = 3x10-5 (bottom panel). D Lack of correlation between MIAA and VEGF-D plasma measures (Spearman's rs n.s., not significant). E Overabundance of MIAA in LAM patients not treated with rapamycin relative to rapamycin-treated (number (n) of samples are indicated). The asterisk indicates significant difference based on two-sided Mann-Whitney test (P = 0.043). Average values are indicated with lilac-colored lines. F Receiver operating characteristic (ROC) curves and the corresponding area under the curve (AUC) and Akaike information criterion (AIC) values for the analyzed metabolites plus VEGF-D, comparing LAM and healthy (left) or related pulmonary disease (right) plasma. G Plasma levels of three other metabolic products derived from monoamine metabolism and showing significant differences from controls and/or between LAM and related pulmonary diseases (individual samples indicated in panel C). The asterisks indicate significant differences based on two-sided Mann-Whitney tests (*P < 0.05, **P < 0.01, and ****P < 0.0001; 4-HPA, control-LAM P = 0.024 and LAM-other P = 0.089; HVA, control-LAM P = 0.008 and LAM-other P = 0.013; and VMA, control-LAM P = 1x10-4 and LAM-other P = 0.10). Average values are indicated with lilac-colored lines. Comparison of the three groups: Kruskal-Wallis test; 4-HPA P = 0.095; HVA P = 0.013; and VMA P = 0.005. H Left panel, concise representation of the mitochondrial reactions mediated by MAOs and ALDHs from a monoamine (R-NH2), to an aldehyde (R-CHO), to the four identified metabolites (R-COOH). The main monoamine origin of each metabolite is indicated in parentheses. Right panel, detail of the histamine metabolism pathway, with enzymes or enzymatic activities marked in red: ALDH, diamine oxidase (DAO), histidine decarboxylase (HDC), histamine N-methyltransferase (HMT), imidazole acetic acid-phosphoribosyl transferase (IPRT), and MAO-B.