EMBO/sd-character-level-ner huggingface dataset:
Figure 4A Expression of HFR1 and protein levels of HFR1-3xHA in seedlings of hfr1>pAt:ChHFR1 (line #22) and hfr1>pAt:AtHFR1 (line #13). Seedlings were grown for 7 days in continuous W (~20 µmol m-2 s-1) after which they were incubated for 3 h in high W (~100 µmol m-2 s-1) and then either kept at high W or transferred to W+FR (R:FR, 0.06) for 3 or 6 h, as indicated in the cartoon at the top. Relative HFR1 transcript levels, normalized to UBQ10, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 #22 grown for 3 h under W+FR. Relative protein levels, normalized to actin, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 #22. Samples were collected at data points marked in the cartoon with asterisks. B Cartoon of constructs containing ChHFR1 or AtHFR1 under the 35S promoter used for transient expression of transgenes in N. benthamiana leaves. C Relative HFR1 transcript levels transiently expressed in tobacco leaves, normalized to the GFP, are the means ± SE of three independent biological replicates (left). Relative HFR1 protein levels, normalized to the GFP levels, are the means ± SE of four independent biological replicates (right). In A and C, asterisks mark significant differences (Student t-test: * p-value <0.05, ** p-value <0.01) between the indicated pairs. D Degradation of ChHFR1 (35S:ChHFR1) and AtHFR1 (35S:AtHFR1) in tobacco leaf discs treated with cycloheximide (CHX, 100 µM) for the indicated times. Tobacco plants were kept under high W (~200 µmol m-2 s-1) for 3 days after agroinfiltration and then leaf circles were treated with W+FR (R:FR, 0.2) and CHX. Relative HFR1 protein levels (ChHFR1, blue bars; AtHFR1, red bars), normalized to the GFP levels, are the means ± SE of four biological replicates relative to data point 0, taken as 1 for each line. Asterisks mark significant differences (2-way ANOVA: * p-value <0.05) between ChHFR1 and AtHFR1 at the same time point.
Crystal structure of the yNap1–H2A–H2B complex.Figure 2A Domain architecture of yNap1, H2A and H2B. The Histone Binding Region (HBR1 and HBR2) of yNap1 and the yNap1 Binding Region (NBR1 and NBR2) of H2A are indicated. Regions involved in higher-order oligomerization are shown (IF1 and IF2). B Ribbon view of the asymmetric crystallographic unit containing a (yNap1c2-H2AΔ14-H2BΔ28)6 assembly. For clarity only layer 1 of the complex is shown. Layer 1 contains three protomers A-C that are related by two perpendicular non-crystallographic dyads ().C Side-view of the complex. The layer 2 omitted from panel B is shown in grey. The yNap1 dimer is shown in green and yellow, H2A in red and H2B in blue. See also Fig EV1.D View of the yNap1-H2A-H2B protomer complex with labeling of secondary structure elements.
Distinct substrate recruitment modules are required to ubiquitinate HBP1 and ZMYND19 in vitro.Figure 2A. Coomassie-stained SDS-PAGE showing purified hGID sub-complexes used for in vitro ubiquitination assays. The catalytic core composed of MAEA, RMND5a and TWA1 is colored in blue, WDR26 in dark cyan, RanBP9 in light magenta, ARMC8 in dark red and GID4 in orange. B. Coomassie-stained SDS-PAGE showing the purified hGID substrates, HBP1 and ZMYND19. C., D. Western blot analysis of in vitro ubiquitinated HBP1, which was performed by mixing purified HBP1 with ubiquitin E1, UBCH5a, and ubiquitin in the presence of the indicated hGID sub-complexes (n=3). E. Immunoblots of in vitro ubiquitinated ZMYND19, which was performed by mixing purified ZMYND19 with ubiquitin E1, UBE2H, ubiquitin and the 6-subunit hGID complex (ARCM8, RanBP9, WDR26, MAEA, RMND5a and TWA1) in the presence or absence of GID4 and a 10-fold excess of the PGLV GID4-specific peptide (n=2) F. Comparison of the N-terminal sequences of the first five amino acids of the Pro-/N-degron consensus motif (Dong et al. 2020) and human ZMYND19 (Q96E35).
Figure 6A. Schematics and immunoblot of LSD1 co-immunoprecipitation (coIP) from PeTa cells. IB, Immunoblot; MS, mass spectrometry. B. Volcano plot displaying the protein-protein binding partners of LSD1. Proteins belonging to the LSD1 complex are marked in red, protein localization in blue and other protein interaction partners in green. FC, fold-change. C. Protein-protein interaction (PPI) mapping of the identified LSD1 binding partners. Individual complexes and p-values are displayed in Fig EV 6A. D. Volcano plot depicting the protein-protein binding partners of LSD1 depleting in PeTa cells treated with ORY-1001 versus DMSO. Proteins belonging to the LSD1 complex are marked in red, protein localization in blue and other protein interaction partners in green. FC, fold-change. E. Violin plot depicting the HMG20B dependency in MCC compared to cancer types from 23 tissues, ordered according to mean. Red horizontal line depicts the median. Data obtained from DepMap RNAi screen. Blood, hematopoietic and lymphoid tissue; U. aerodigestive, upper aerodigestive tract; A. ganglia, autonomic ganglia; CNS, central nervous system. F. Schematic of TMT-MS experiment. PeTa cells were treated for 24h with 100 nM GSK-LSD1 or DMSO. Cells were fractionated to separate nuclei from cytosol. Nuclei were analyzed by TMT-MS. IB, Immunoblot; TMT-MS, tandem mass tag labeling mass spectrometry. G. Volcano plot depicting the differentially regulated proteins obtained from Fig 6F. Proteins members of the LSD1-CoREST complex are labeled and highlighted in red. Proteins significantly upregulated (-log10(p-adjusted)≥1; log2FC≥0.75) and involved in nervous system development (GO:0007399) are labelled. FC, fold-change. H. Left. Immunoblot confirmation of HMG20B downregulation. H3 serves as nuclear fraction control and α-Tubulin as loading control. Right. Immunoblot quantification. HMG20B protein levels are normalized to H3 and relative to DMSO control. I. Amino acid conservation analysis of HMG20B across species and (bottom) the corresponding domains. DNA interacting amino acids are indicated with a red dot. aa, amino acid; NLS, nuclear localization signal (aa: 55-65); HMG, high-mobility group box (aa: 70-137); cc, coiled-coil domain (aa: 196-251); ALPHA, alpha-helices (aa: 276-312). J. Schematic of HMG20B domain deletion mutant proteins. WT, wild-type; ΔALPHA, alpha-helices deletion; ΔCC, coiled-coil domain deletion; ΔHMG, HMG box deletion. K. Bar graph of HMG20B rescue experiment in PeTa cells transduced with either a shRNA targeting HMG20B (shHMG20B) or negative control (shRenilla) and with an overexpression construct expressing either of the HMG20B mutant that cannot be targeted by the shRNA. Cell survival is depicted at day 8 after transduction and normalized to shRenilla. EV, empty vector control, WT, wild-type; ΔALPHA, alpha-helices deletion; ΔCC, coiled-coil domain deletion; ΔHMG, HMG box deletion.
Figure 3(A) Two views of C. glabrata Hrr25 (Apo form) with domains colored as in Figure 1, showing bound PO4- ions and the previously-identified Mam1-binding residues His25 and Glu34 (Petronczki et al, 2006). (B) Two views, roughly equivalent to the views in (A), of the Hrr25 central domain, colored as a rainbow according to the schematic at top. Dotted lines in the schematic indicate disordered loops. See Figure EV1A for sequence alignment of this domain.
Reduced angiogenesis and cardiomyocyte proliferation in CM-G4-KO mice.Figure 2(A) Representative pictures of TUNEL, WGA (wheat germ agglutinin to mark cardiomyocyte membranes) and DAPI immunofluorescence staining of mouse hearts as indicated. The white dashed line encircles the infarcted area (arrows). Scale bar: 200µm. The quantification of the area of TUNEL positive cells 3 hours after cryoinjury is shown on the right (A´). The number within bars indicates the number of mice analyzed in that particular group. (B) Representative merged and single channel pictures of cardiac CD31, DAPI and WGA immunostaining in mouse hearts as indicated. The quantification of the myocardial capillary density as capillaries/cardiomyocyte (CM) ratio, 7 days (d) after cryoinjury or sham surgery is shown as bar graph (B´). Scale bar: 20µm. **p=0.0039. (C) Representative pictures of myocardial immunofluorescence staining for the mitosis marker phospho-histone H3 (pH3), troponin T and DAPI in mice as indicated. Enlarged pH3 positive cardiomyocyte nuclei are shown separately. The arrows indicate pH3 positive cardiomyocyte nuclei. Scale bar: 25µm. The quantification of pH3 positive (+) cardiomyocytes per microscopic field is shown as bar graph (C´); *p=0.046. (D) Representative myocardial immunostaining for aurora B, DAPI and troponin T (TropT). The arrow points towards a cardiomyocyte cell division, in which aurora B localizes at the midbody in cytokinesis. Scale bar: 25µm. The quantification of dividing cardiomyocytes per microscopic field is shown as bar graph (D´); *p=0.0104. (E) Determination of cardiac gene-expression by qPCR 7 days after sham or cryoinjury for the indicated genes; *p=0.039 for Il13 and p=0.049 for Vegfa; **p=0.0043 for Ccne1, p=0.0027 for Cdk4, p=0.0096 for Cenpa, p=0.0015 for Zfp191; ***p=0.0009 for Ccna2, p=0.0002 for Cenpa.
Induction of macroautophagy in L/APP, SH-SY5Y, and N2a cells.Figure 4.Induction of macroautophagy in L/APP, SH-SY5Y, and N2a cells. (A and B) EM images showing changes in the number of AVs (arrows) in L/APP-overexpressing APP695 (L/APP cells) grown in complete medium (A, top left) or in medium lacking Leu and His (A, top right) for 6 h and in SH-SY5Y cells grown in the presence (A, bottom left) or absence (A, bottom right) of serum. At higher magnification, early and late AVs with typical morphologies are seen in a Leu/His-deprived L/APP cell (B). (C) Fluorescent and immunofluorescent labeling of large vesicles by the AV marker monodansylcadaverine (0.5 μg/ml for 30 min; left) and LC3 antibody (middle, L/APP; right, SH-SY5Y) in macroautophagy-induced cells (bottom), which are much less abundant in cells grown in complete medium (top). (D) Western blots confirm the cytochemical evidence for increased LC3-II levels as well as phospho-mTOR (P-2481) but not total mTOR after macroautophagy induction by Leu and His deprivation or 10 nM rapamycin (Rap) and macroautophagy inhibition by 5 mM 3MA in L/APP, N2a, and SH-SY5Y cells. Immunoblots for LC3 in SH-SY5Y cells and P-2481 mTOR in L/APP cells have been spliced but are derived from the same blot.
fdsc-αDEC localizes with TLR9 in DCs.Figure 5A-D BMDCs from C57BL/6mice were stably transduced with YFP-tagged TLR9. Cells grown on coverslips were then incubated with TRITC-conjugated wild-type (A, B) or fdsc-αDEC (C, D) bacteriophages for 6 h. Cells were then washed, fixed and analyzed by confocal microscopy. Nuclei were counterstained with Hoechst. (B, D) are enlargements of a single cell. Representative images are shown. Numbers at bottom of right panels indicate the percentage of TLR9bacteriophage co-localization (mean values ± SD, N = 2, n = 100). Scale bars, 10 mm.
Figure 1Volcano plot highlighting proteins significantly enriched (open circles, FDR<0.05) in eNOS-FLAG immunoprecipitates from growth factor-stimulated human endothelial cells; n=3 independent cell batches. Nitrite in the cell supernatant of HEK293 cells expressing wild-type (WT), Y657F (YF) or Y657D (YD) eNOS and treated with solvent (Sol) or ionomycin (Io, 1 µmol/L, 15 min); n=6 independent experiments (2-way ANOVA and Bonferroni). The FMN/FAD ratio measured in eNOS immunoprecipitates from cells expressing WT, YF or YD eNOS; n=6 independent experiments (1-way ANOVA and Newman-Keuls). Consequence of mutating Y657 on the growth factor-induced formation of eNOS complexes with Hsp90 and calmodulin (CaM); n=6-19 independent cell batches (1-way ANOVA and Newman-Keuls). Effect of mutating Y657 on the formation of complexes between eNOS, Hsp90 and PKM2 in growth factor-stimulated human endothelial cells. Complex formation was assessed in eNOS-FLAG immunoprecipitates (E), or PKM2 immunoprecipitates (F); n=4-8 independent cell batches (1-way ANOVA and Newman-Keuls).
Figure 2Schematic showing the difference between two isoforms of AGO1. Detection of endogenous Ago1x by Western blot in HEK293 cells using anti-Ago1x antibody and pre-immune serum. Western blot of lysates from HEK293 and HeLa cells using anti-Ago1x antibody pre-treated with Ago1x-specific peptide. Expression of Ago1x in HEK293 cells transfected with three AGO1-specific siRNAs (1,2,3). qRT-PCR analysis of AGO1 transcript expression in these cells is shown below. Proportion of Ago1x isoform in HEK293 cells. To separate Ago1x and Ago1 (~ 3 kDa difference), the cell lysate was electrophoresed for 3.5 h in 10% polyacrylamide gel. Proportion of Ago1x and canonical isoform (Ago1) was calculated by densitometric analysis. Results from three independent experiments are shown as a bar graph (mean ± S.E.). Expression of Ago1x in mouse organs. Presence of ribosome footprint in the Inter stop codon region of AGO1 transcript in U2-OS cells. Ribosome profiling data was taken from NCBI's sequence read archive (SRA Accession no. SRR1257257) and ribosome footprints on AGO1 transcript were analyzed as described in Methods section. Zoomed area near the inter-stop codon region is shown in the inset. Position of canonical stop codon and the downstream in-frame stop codon are shown. Detection of Ago1x by Western blot in U2-OS cells is shown (right).
Figure 4A. Experimental approach used for the identification of pseudouridine sites in mitochondrial RNA. A combination of the CMC modification and the next-generation sequencing on pure mitochondrial RNA from control and siRNA treated cells was used. CMC-ψ modification causes skips in reverse transcription rather than stops, resulting in 1bp deletions in sequence alignments. B. Quantification of Δ (the difference between the rate of deletions in CMC treated vs un-treated cells) for individual ψ sites in control and siRNA treated cells. The bars represent the mean ± SEM of two independent experiments. P-values were calculated using a one-tailed t-test (* P<0.05; ** P<0.01; ***P<0.001).
Wnt7a increases TNTs and vesicle transfer in CAD cells.Figure 1Representative confocal images of CAD cells exposed for 4 h to the indicated treatments. Cells were fixed and then stained with WGA (green) and DAPI (blue). Percentage of TNT-connected CAD cells with each of the indicated treatments. Top: Schematic of the co-culture system used to assess vesicle transfer from donor (labeled with DiD) to acceptor (labeled with CTG) cells. After labeling, cells were detached, replated in 1:1 ratio and cultured in control conditions or treated with 200 µM H2O2 or 200 ng/mL Wnt7a for 4 h. Middle: Representative confocal images of co-cultured donor and acceptor CAD cells. Each image corresponds to the projection of the entire Z-stack. Bottom: The insets correspond to the projection of selected Z-stacks of the area marked with a yellow square in each condition to better illustrate the transferred vesicles in acceptor cells. Yellow arrows point to DiD-labeled vesicles inside acceptor cells. Percentage of acceptor cells containing DiD-labeled vesicles in the co-culture systems with the indicated treatments. Top: Schematic of the culture system used to assess vesicle transfer by secretion. Donor cells (labeled with DiD) were cultured in control conditions or treated with 200 µM H2O2 or 200 ng/mL Wnt7a for 4 h. Then acceptor cells (labeled with CTG) were treated for 4 h with the conditioned media from donor cells. Bottom: Representative confocal images of acceptor CAD cells treated with the conditioned media from donor cells. Yellow arrows point to DiD-labeled vesicles inside acceptor cells. Percentage of acceptor cells containing DiD-labeled vesicles after being cultured for 4 h with the conditioned media of treated donor cells.
a) Intracellular ROS production was assessed using DCFH-DA.f3(a) Intracellular ROS production was assessed using DCFH-DA. (b) Plasma membrane calcium influx was measured by analysing the quench of Fura-2 fluorescence upon addition of extracellular Mn2+. (a,b) Were conducted in enzymatically digested single FDBs from WT, mdx and p47−/−-mdx mice. Bars represent average±s.e.m. from n=15 individual fibres for each condition. (c) Immunoblot analysis of Src protein with anti-P-Src or anti-Src antibodies. (d) Autophagy marker proteins were analysed by immunoblotting with antibodies as indicated (c) and (d) were conducted in enzymatically isolated fibres from FDBs of WT, mdx and p47−/−-mdx mice. Representative images are shown. GAPDH was detected as a loading control. Bars represent average±s.e.m. from n=3 independent biological experiments. (e) LC3-LAMP1 colocalization in single FDBs from mdx and p47−/−-mdx mice was analysed using confocal microscopy. Representative images from n=3 independent biological experiments are shown (scale bar=100 and 40 μm for red box areas). f) Immunofluorescence assay to detect lysosomes (LAMP1) in TA muscle from WT, mdx and p47−/−-mdx mice. White arrows indicate lysosome and yellow arrows indicate nucleus. (g) Immunohistochemistry to detect lysosome (LAMP1) in TA muscle from WT, mdx and p47−/−-mdx mice. Black arrows indicate LAMP1 positive (brown) structures. Histogram plot quantifying the number of immunopositive LAMP1 structures per fibre. Representative images from n=3 independent biological experiments are shown. Scale bar represents 100 μm for f and 140 μm for g. Statistical differences between groups were determined using ANOVA with Tukey's post hoc test. *P0.05 and **P0.01.
Xenophagy in oropharyngeal keratinocytes can be stimulated by SLO or SLS and is not associated with killing of intracellular GAS.Figure 2A. Confocal microscopy of the association between EGFP-LC3 and GAS strain 188, 188SLO- (SLO-), 188SLS- (SLS-), or 188SLO-SLS- (SLO-SLS-). Immunofluorescent staining distinguished intracellular (Alexa-568, red) from extracellular (Alexa-568 and Alexa-660, red and blue, respectively) GAS. Scale bar = 10 µm. The percent of intracellular GAS that were associated with EGFP-LC3 at 3 h post-infection is shown for each strain. B. Electron microscopy of GAS strains 188, 188SLO-, and 188SLO-SLS- at 3 h post-infection. Arrowheads indicate the presence of double or multiple membranes (188 and 188SLO-) or single membranes (188SLO-SLS-) partially or completely surrounding bacteria. Vacuolar compartments associated with 188 and 188SLO- also contain cytosolic material. C. Intracellular survival of GAS strains 188, 188SLO-, 188SLS-, and 188SLO-SLS-. *, P<0.05. D. Intracellular survival of 188 or 188SLO- in the presence or absence of Beclin1 knockdown.
Figure 1(a) Scheme of the shRNA-mediated synthetic lethal screen. Huh7-IR and CR cells were infected with lentiviral vectors (MOI=0.5) expressing the shRNA library (human genome-wide pooled lentiviral shRNA library module 1, Vector: pRSI16cb, Cellecta) and cultured with 2μg/ml Puromycin for the selection of shRNA-expressing cells plus 7μM Sorafenib (Srf). After 4 weeks of culture, genomic DNA was extracted, and shRNA barcodes were amplified for next-generation sequencing to uncover the critical genes for Sorafenib resistance. (b) Combinatorial analysis of genes differentially expressed between Sorafenib-sensitive and resistant cells and genes with depleted barcodes in the synthetic lethal screen in Huh7-IR and CR cells. 38 common genes were identified, among which was WWTR1 coding for TAZ. (c) Huh7-parental, IR and CR, and Hep3B-parental, and IR and CR cells were treated with different concentrations of Sorafenib (0, 3, 6, 9μM) for Huh7-P/IR/CR and Sorafenib (0, 2, 4, 6μM) for Hep3B-P/IR/CR for 18 hours before harvest. Protein levels of YAP and TAZ were determined by immunoblotting illustrating higher protein levels of YAP/TAZ in Sorafenib-resistant cells. GAPDH served as loading control. Results represent three independent experiments. (d) Colony formation assay showing that shRNA-mediated depletion of YAP/TAZ leads to cell numbers in response to Sorafenib treatment. Huh7 IR and CR cells either expressing a control shRNA (shLuc, non-targeting shRNA) or shRNA against both YAP and TAZ (shY/T) were treated with different concentrations of Sorafenib (0, 4, 8μM) for 2 weeks and colonies were visualized by crystal violet staining. Results represent three independent experiments. (e) Gene Set Enrichment Analysis (GSEA) of the genes differentially expressed between YAP/TAZ-deficient (siY/T) and control siRNA (siCtrl) transfected HLE cells showed an enrichment for genes involved in the regulation of lipid peroxidation. (f) Basal reactive oxygen (ROS) levels increased upon loss of YAP/TAZ. HLE-shLuc and HLE-shY/T cell lines were stained with CellROX™ Green Flow Cytometry Assay Kit, and ROS levels were measured by flow cytometry using a 488 nm laser. Results represent three independent experiments. (g) Basal lipid peroxidation levels increased with the loss of function of YAP/TAZ. HLE-shLuc and HLE-shY/T cells were stained with C11-BODIPY 581/591. Reduced-Bodipy was measured by flow cytometry using a 488 nm laser, and oxidized-Bodipy was measured with a 561 nm laser. A significant shift of oxidized-Bodipy occurred upon depletion of YAP/TAZ. Results represent three independent experiments. (h) Colony formation assay demonstrating that the ferroptosis inhibitor Ferrostatin-1 (Fer) reversed Sorafenib-induced cell death in YAP/TAZ-deficient HCC cells. HLE-shLuc and shY/T cells were treated with different concentrations of Sorafenib (0, 2, 4μM) and either DMSO or Ferrostatin-1 (Fer; 5μM) for 2 weeks. Results represent three independent experiments.
Figure 2A) Distributions of brain size as indexed by total brain volume (TBV) in ASD and control toddlers used in the co-expression analysis (WGCNA). T= value from T-test; F= value from Levene's test. B) Module eigengenes (MEs) from the combined WGCNA analysis are correlated with TBV measures in all brains, ASD and control groups. P-value is in parenthesis and adjusted p-value (q-value) is <0.05 for all seven modules. Significant associations after 10000 permutation tests are provided in Figures E4 and E5. C) Metacore enrichment scores of the seven (7) modules initially related to brain size variation across all subjects. Each module is called by its assigned color and represents the top process network obtained by the enrichment analysis in Metacore GeneGO (see also EV1). D-F) (i) Linear modeling of Module Eigengenes (MEs) by TBV measures in control (blue) and ASD (red) toddlers. p-value **< 0.01; cor= correlation coefficient; ns= not significant. See also Figure 2B for cor and p-values. (ii) Linear modeling of GS by GC to display changes in network organization relevant to brain size. Significance codes: p-value ***<0.001; **< 0.01; *<0.05; cor= correlation coefficient; ns= not significant. (iii) The top 30 genes with highest values for GS and GC were compared between ASD and control. Purple indicates the number of genes that moved away from the top 30 rank position between the two groups (Different genes) and grey indicates the number of genes that did not (Common genes).
Figure 1(A) Protein abundance is the normalized signal intensity (LFQ) for a protein divided by its molecular weight. Specificity (enrichment) is the ratio of the protein LFQ intensity in the MIA40FLAG fraction to control samples. The LFQ for proteins that were not detected in the control samples was arbitrarily set to 1 for calculation purposes. LFQ, Label-Free-Quantification; M.W., molecular weight. (B) Flp-In T-REx 293 cells with induced MIA40FLAG expression were solubilized, and the affinity purification of MIA40FLAG was performed. Fractions were analyzed by SDS-PAGE and Western blot. Load: 2.5 %. Eluate: 100 %. Unbound: 2.5 %. (C) Flp-In T-REx 293 cells with induced MIA40FLAG expression were solubilized, and the affinity purification of MIA40FLAG was performed. Eluate fractions were solubilized under reducing (DTT) or non-reducing (IAA) conditions and analyzed by SDS-PAGE and Western blot. DTT, dithiothreitol; IAA, iodoacetamide. Eluate: 100 %. (D) Cellular protein extracts from Flp-In T-REx 293 cells with induced expression of wildtype or mutant MIA40FLAG were subjected to affinity purification. Load and eluate fractions were analyzed by reducing SDS-PAGE and Western blot. Load: 2.5 %. Eluate: 100 %.