EMBO/sd-character-level-ner huggingface dataset:
Figure 4A Compartments of N-Hcy substrates identified by proteomic survey were predicted by WoLF PSORT. B WebLogo visualization of the amino acids spanning N-Hcy-modified lysine sites. C SOD activities were down-regulated by in vitro N-homocysteinylation. Flag-tagged SOD1, SOD1K23W,K122W,K128W (3KW) mutant, SOD2, and SOD2K44W, K51W,K98W,K106W,K178W (5KW) mutant were expressed in HEK293T cells, affinity purified, and incubated with or without 10 μM HTL in solution (in vitro). After 12 h, the reaction mixture was desalted and concentrated with filter tubes. N-Hcy levels and SOD activities were determined and normalized to untreated WT SOD1 and SOD2, respectively (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons. D Upon switching N-Hcy substrate lysine (K) sites to nonmodifiable tryptophan (W), the N-Hcy unmodifiable mutants displayed lower specific activity than the wide-type (WT) SOD. SOD1 (WT, K23W, K122W, K128W, and 3KW) and SOD2 (WT, K44W, K51W, K98W, K106W, K178W, and 5KW) were expressed in HEK293T cells. The catalytic activity of affinity-purified SOD proteins was determined and normalized to protein levels. Wild-type SOD1 and SOD2 activities were set at 100% (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons. E, F Hcy and HTL treatment in NE4C cells increased N-homocysteinylation of SODs and decreased their activities. Flag-tagged SOD1 and SOD2 were each ectopically expressed in NE4C cells. The cells were transferred to serum-free DMEM with or without 20 μM Hcy (E) or 10 μM HTL (F) 6 h before harvesting. After affinity purification, the specific activities of SOD1 and SOD2 were determined and normalized to SOD1 and SOD2 activity in untreated cells. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons. G N-Hcy SOD levels were down-regulated in MARS/MARS2 knockdown cells. Flag-tagged SOD1 and SOD2 were each over-expressed in untreated and MARS/MARS2 knockdown NE4C cells. After affinity purification, the N-Hcy levels and relative specific activities of purified SOD1 and SOD2 were determined and quantified relative to the NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.
Figure 3PC9 cells (exon 19 deletion) were subcutaneously implanted in the flank of CD1-nu/nu mice (3X106/mouse). When tumors became palpable, mice were randomized in groups of 5-9 animals and treated for 90 days (grey areas) with 2XmAbs (cetuximab plus trastuzumab, each at 0.1 mg/mouse/injection) once every three days, or daily with different TKIs: osimertinib (5 mg/kg), afatinib (5 mg/kg) or imatinib (100 mg/kg), either alone or in combination with the two antibodies. Following 60 days of treatment, the frequency of TKI administration was reduced to once every other day (underneath dotted lines), while mAb treatment remained unaltered. Tumor growth (A) are shown. Mice were euthanized when tumor size reached 1,500 mm3. PC9 cells (exon 19 deletion) were subcutaneously implanted in the flank of CD1-nu/nu mice (3X106/mouse). When tumors became palpable, mice were randomized in groups of 5-9 animals and treated for 90 days (grey areas) with 2XmAbs (cetuximab plus trastuzumab, each at 0.1 mg/mouse/injection) once every three days, or daily with different TKIs: osimertinib (5 mg/kg), afatinib (5 mg/kg) or imatinib (100 mg/kg), either alone or in combination with the two antibodies. Following 60 days of treatment, the frequency of TKI administration was reduced to once every other day (underneath dotted lines), while mAb treatment remained unaltered. animal survival (B) are shown. Mice were euthanized when tumor size reached 1,500 mm3. (C) Statistical analysis of tumor volumes corresponding to the last measurement for each mouse was performed using one-way ANOVA followed by Tukey's multiple comparison test. (D) Shown are tumor volumes corresponding to individual animals of each group. The numbers of mice with undetectable tumors are indicated. The grey areas represent treatment phases and they are followed by drug holidays (blank).
Figure 4A Generation of two target site blocker (TSB) locked nucleic acid sequences: TSB1 and TSB2 (in blue), for the disruption of the miR-34a/Pdgfra interaction, indicating binding sites in the Pdgfra 3′-UTR, Kit 3′-UTR, and the Sirt1 3′-UTR (in black), alongside the miR-34a sequence (in red). The miR-34a seed sequence, and the seed-sequence binding site in the target mRNA 3′-UTR are indicated in bold, and brown, respectively. B Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR (SCR) and miR-34a (MIM34a) mimics, and probing for PDGFRα and c-Kit as TSB‑dependent and TSB‑independent target readouts, respectively. Protein loading equivalence was controlled by βactin levels. Note: PDGFRα, βactin, and c-Kit were all probed on the same membrane; hence a single βactin immunoblot is presented. Data are representative of three experiments. C Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR (SCR) and miR-34a (MIM34a) mimics, and probing for PDGFRα and SIRT1 as TSB‑dependent and TSB‑independent target readouts, respectively. Protein loading equivalence was controlled by βactin levels. Note: PDGFRα, βactin, and SIRT1 were all probed on the same membrane; hence a single βactin immunoblot is presented. Data are representative of three experiments. D Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type mouse pups at post-natal day (P) 14, treated with either scrambled target site blocker (SCR) or a cocktail of both target site blockers (TSB1 and TSB2) during normal (21% O2) and aberrant (85% O2) alveolarization (scale bar, 50 µm). Data are representative of three or more experiments. E Quantification of total number of alveoli by design-based stereology in wild-type mouse pups at P14, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group). F Quantification of mean septal thickness by design-based stereology in wild-type mouse pups at P14, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group). G Quantitative analysis of PDGFRα+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group). H Quantitative analysis of PDGFRα+/αSMA+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).
Figure 8(A) Overexpression of Pin2 enhances Chs5 stabilization at the TGN. Binding kinetics of Chs5 GFP in WT, bud7bch1 and bud7bch1 strain expressing Bch2-dependent cargo Pin2 from a centromeric or a 2μ plasmid. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2. (B) Comparison of the expression levels of the endogenous Pin2 with the levels of Pin2 expressed from a centromeric or a 2μ plasmid. Pgk1 serves as loading control. A representative immunoblot of three independent biological experiments is shown.
CY exhibited activity with CLL samples.Figure 5Dose curve of CY, fludarabine, and bendamustine using fresh CLL samples. Data represent mean ± SD. CY preferentially killed CLL cells over healthy B cells. Data represent mean ± SD Toxicity of CY190602 in patients harboring deletion of chromosome 17p (including p53 locus) compared to patients without del17p. Viability of samples was normalized to background apoptosis for individual patient samples. For all experiments, cell viability was determined after 72 h of drug treatment. Data represent mean ± SD.
Figure 5(A) Persistence of γH2AX- and MDC1-positive foci in absence of NES-phosphorylation or RG-methylation of separase. Transfected Hek293T cells were siRNA- and Dox-treated to deplete endogenous separase and induce expression of the indicated, siRNA resistant Myc-tagged separase variants, respectively. Then, they were constitutively (− washout) or transiently exposed to DRB (+ washout) and finally quantitatively assessed by IFM for γH2AX- and MDC1-positive foci. Shown are averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each. Scale bar = 5 μm  (B) Schematic of the experiment  (C) Recruitment of separase-WT and -S1660D but not separase-superNES, -S1660A, and -KG to DSBs. Cells from (A) were treated as indicated and analysed by IFM for co-localization of Myc-separase with γH2AX-foci. Scale bar = 5 μm  (D) Interaction of separase-WT and -S1660D but not separase-superNES, -S1660A, and -KG with γH2AX. Cells from (A) were subjected to (IP-)Western analysis as indicated. Lanes that illustrate cellular levels of cyclin A2 and γH2AX after DRB-washout and those that analyse interaction of Myc-separase variants with γH2AX in presence of DRB are labeled by red arrows and blue arrow heads, respectively
Figure 1A Schematic illustration of intranasal delivery of AAV into the MOE. B The expression of mature miR-200b and miR-200a in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; *p<0.05, **p<0.01; Student's t-test). C The expression of Ttll10 (the host gene of miR-200b/a) mRNA in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; p=0.3540; Student's t-test). D The locomotor activity of the miR-200b/a KD mice was similar to that of the NC mice (NC: n=13 mice, miR-200b/a knockdown: n=15 mice; data represent the mean±SEM; p=0.5030; Student's t-test). E The odorant habituation results shown as the ratio of odor sniffs/water of the miR-200b/a KD mice were significantly reduced compared to those of the NC mice. These odorants included male urine (1:50), female urine (1:50), isopentyl acetate (50 μM), citral (50 μM), 2-heptanone (50 μM) and propyl propionate (50 μM) (n=6 mice each group; data represent the mean±SEM; *p<0.05, **p<0.01, ***p<0.001; Student's t-test). The resident/intruder experiment showed that the attack frequency (F) of the miR-200b/a KD mice were impaired compared to those of the NC mice (NC: n=11 mice, miR-200b/a KD: n=10 mice; data represent the mean±SEM; ****p<0.0001; Student's t-test). G The resident/intruder experiment showed that the attack duration (G) of the miR-200b/a KD mice were impaired compared to those of the NC mice (NC: n=11 mice, miR-200b/a KD: n=10 mice; data represent the mean±SEM; ****p<0.0001; Student's t-test). H, I The analysis of male-female mating behaviors showed that the mating frequency (H) and duration (I) of the miR-200b/a KD mice were significantly reduced compared to those of the NC mice (NC: n=11 mice, miR-200b/a KD: n=10 mice; data represent the mean±SEM; **p<0.01, ***p<0.001; Student's t-test).
Co‐localization of Hsp70 with Siglec‐5 and Siglec‐14 ligands in lung bronchiolesImmunofluorescence of human lung and prostate sections probed with Siglec‐5 R119A Fc or Siglec‐14 R119A Fc (red) with an anti‐Hsp70 antibody (green).Figure 2Co‐localization of Hsp70 with Siglec‐5 and Siglec‐14 ligands in lung bronchiolesImmunofluorescence of human lung and prostate sections probed with Siglec‐5 R119A Fc or Siglec‐14 R119A Fc (red) with an anti‐Hsp70 antibody (green). The co‐localized signals for the Siglec‐Fc probes with an anti‐Hsp70 antibody suggest that Hsp70 is one of the possible sialic acid‐independent ligands of Siglec‐5 and Siglec‐14 in the lung bronchiole. White dashes mark the lung bronchiole epithelium. All images are representative of at least 2-3 replicates.
Figure 2Knockdown of GR in BAT assessed by immunoblotting. Β-Tubulin was used as loading control. Quantification of immunoblot in (A) using Image Lab (n = 6 animals per group). Body temperature over the course of the cold exposure, determined daily (n = 6 animals per group). Body weight at the end of the cold exposure (n = 6 animals per group). BAT weight at the end of the cold exposure (n = 6 animals per group). Average daily food intake in g/day (n = 6 animals per group). Representative Hematoxilin and eosin staining image of BAT of cold-exposed Flox and GRBATKO mice. Scale bar 50 µm. Triglyceride content in BAT from Flox and GRBATKO animals (n = 5-6 animals per group). Heatmap of mean gene expression fold changes in thermogenic marker genes based on BAT microarray analysis comparing GRBATKO vs Flox control animals, indicating unchanged thermogenic marker gene expression. Color gradient ranges from 2-fold higher expression in GRBATKO over Flox controls (red) to 0.1-fold (lower) expression (blue) (n = 4 animals per group). Volcano plot from microarray gene expression data as in (I), with cut-off values for the adjusted p-value <0.1 and fold change >1.5. Serum corticosterone levels (n = 6 animals per group).
Scl binding to cardiac enhancers occurs transiently in mesodermVenn diagram showing overlaps of Scl binding sites in different developmental stages—Flk1+ MES, fetal liver (FL) erythroblast cells (Kassouf et al, ) and hematopoietic progenitors (HPC7) (Wilson et al, ) (left)—shows that majority of Scl binding sites are developmental stage specific.Figure 3A Venn diagram showing overlaps of Scl binding sites in different developmental stages-Flk1+MES, fetal liver (FL) erythroblast cells (Kassouf et al, ) and hematopoietic progenitors (HPC7) (Wilson et al, ) (left)-shows that majority of Scl binding sites are developmental stage specific. Examples of developmental stage‐specific Scl‐bound genes are shown (right). B GREAT (McLean et al, ) analysis of significantly enriched GO terms in Mouse Phenotype category for all common, Flk1+MES‐specific, HPC7‐specific and FL erythroblast‐specific peaks shows enrichment of heart‐related terms only among Flk1+MES‐specific peaks. C ChIP‐seq tracks with Scl binding in Flk1+MES, HPC7, FL erythroblasts and MEL (mouse erythroleukemia cells) around cardiac (Gata4, Myocd, Nkx2.5) and hematopoietic (Gata1, Gata2 and Lyl1) genes show that binding to cardiac genes occurs only in mesoderm, while binding to hematopoietic genes is maintained throughout hematopoietic development. D Comparison of the location of developmental stage‐specific Scl binding sites relative to TSS shows that Scl binding near TSS occurs more often in later hematopoietic development compared to mesoderm. E Heatmaps of combinatorial clustering of H3K4me1 around Sclmesodermal binding sites in MES, HPC7 and MEL reveal two major clusters: Cluster L loses H3K4me1, H3K27ac and Scl binding during hematopoietic differentiation and is enriched for genes involved in heart development; cluster R retains H3K4me1, H3K27ac and Scl binding during hematopoietic differentiation and is enriched for hematopoiesis‐related genes. Selected GO terms among top 10 most enriched are shown.
Figure 3A Sec63-mNeon images of the cortical ER of WT and Δice2 cells harboring the inducible system (SSY1405, 1603). Cells were untreated or treated with 800 nM estradiol for 6 h. B Classification of peripheral ER structures from cortical sections of cells expressing Sec63-mNeon and Rtn1-mCherry as tubules (purple), sheets (green) or tubular clusters (yellow). Tubular clusters are combined with tubules in the final classification, as illustrated by the overlay. C Quantification of peripheral ER structures in WT and ∆ice2 cells harboring the inducible system (SSY1405, 1603) and treated with 800 nM estradiol for the times indicated. Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant. D mRNA levels of the Ino2/4 target gene INO1 upon ino2* expression in WT and Δice2 cells harboring the inducible system (SSY1405, 1603) as measured by quantitative real-time PCR. Data were normalized to untreated WT cells. Mean + s.e.m., n = 3, biological replicates. Asterisks indicate statistical significance compared with the corresponding untreated cells, as judged by a two-tailed Student's t-test assuming equal variance. Exception was the test against the normalized value for wild-type cells, for which a two-tailed Student's t-test with unequal variance was applied. *, P < 0.05; **, P < 0.01. E Quantification of peripheral ER structures in untreated WT, Δice2, Δopi1 and Δice2 Δopi1 cells (SSY1404, 2356, 2595, 2811). Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant.
Figure 5A The senescence phenotype of the vector control, pER8::CDF4 and pER8::CDF4::RNAi detached leaves treated with H2O2 and incubated in the dark for three days. The third leaf in rosettes of 3-week-old plants was detached and incubated in MES buffer (2 mM MES, pH 5.8) treated with 2 mM H2O2 and 20 μM β-estradiol under dark conditions for three days. Picture shows the leaves before and after treatment. Scale bars indicate 1 cm. (B) DAB staining of H2O2 before and after 2 mM H2O2 plus 20 µM β-estradiol treatment under dark conditions for two days. The brown color represents H2O2 accumulation. Scale bars indicate 1 cm. (C) measurement of H2O2 before and after 2 mM H2O2 plus 20 µM β-estradiol treatment under dark conditions for two days. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test. D Chlorophyll concentrations in the leaves shown in A. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.
TMX1 silencing causes elevated ROS via mitochondria and NADPH oxidase 4 A-C Mitochondrial H2O2 (mito-HyPer) was measured in two cell lines upon transient TMX knockdown.Figure 5Mitochondrial H2O2 (mito-HyPer) was measured in two cell lines upon transient TMX knockdown. Exemplary ratiometric images (F505 nm / F420 nm) are shown (A); corresponding quantification for (B) WM3734 and (C) Mel Juso. , data are presented as mean ± SEM (n values: WM3734: control=546, TMX1 kd=510, TMX3 kd=621; Mel Juso: control=416, TMX1 kd=418, TMX3 kd=442). Mitochondrial Ca2+ uptake in WM3734 with stable TMX1 knockdown was measured using a mitochondria-targeted calcium sensor (4mt-D3cpV) (D). Cells were exposed to 0.25 mM Ca2+ containing Ringer's buffer and Tg. (E) Quantification of basal mitochondrial Ca2+ levels and (F) mitochondrial Ca2+ uptake (basal-plateau). , data are presented as mean ± SEM (n values: control=62, TMX1 kds=45). Mitochondrial volume was determined using MitoTracker DeepRed in WM3734 cells with stable knockdown of TMX1. The mean mitochondrial volume was modeled based on microscopy, the quantification of mean volume (µm3) and mean surface (µm2) is depicted in (G) and (H) respectively. data are presented as boxplots (n values: control=101, TMX1 kds=90; center line: median; box: 25 % and 75 % percentile; whiskers: 1.5 times interquartile range; outliers are shown as dots). Peripheral mitochondria were quantified in WM3734 cells with stable knockdown of TMX1. A peripheral mask was applied based on the membrane staining (CellMask Green) and mitochondria covered area (MitoTracker DeepRed) was evaluated (see Fig. 9). Representative images (I) and quantification (J). In (J), data are presented as mean ± SEM (n values: control=163, TMX1 kds=116). Representative electron micrographs of control and 1205Lu cells with stable TMX1 knockdown (K). Corresponding quantification of the distance between (L) mitochondria and plasma membrane, (M) mitochondria and ER, and (N) the length of the MAM (mitochondria-ER contact site). In (L), data are presented as mean ± SEM (n values: control=100, TMX1 kds= 100). In (M-N), data are presented as mean ± SEM (n values: control=60, TMX1 kds= 60). Cellular H2O2 concentration (HyPer) 48 h after transient silencing of TMX1 and / or NOX4 in WM3734 cells or after inhibiting NOX4 with GKT137831 (140 nM) (O) Representative ratiometric images (F505 nm / F420 nm) and (P) quantification. In (P), data are presented as mean ± SEM (n values: control=837, TMX1 kd=888, TMX1 kd + NOX4 kd=793, TMX1 kd + GKT=844).
Correlative analysis between murine experimental and human neovascular CNV.Figure 9a) H2-Aa expression visualized by trajectory analysis and represented in a t-SNE plot. b) H2-Aa immunohistochemistry (green) by Iba1+ (red) microglia inside lesioned and non-lesioned areas in the retina and RPE. Arrows indicate microglia with aquired MHCII expression whereas asterisks mark microglial cells that remained MHCII negative. Picture is representative of two mice. In the RPE, arrow heads label Tom-MHCII+ cells. Scale bars represents 25 µm. c) HLA-DRA mRNA expression examined by RNA-Seq via Massive analysis of cDNA ends (MACE) analysis of human formalin-fixed and paraffin-embedded (FFPE) membranes of choroidal neovascularization (CNV). Bars represent means ± s.e.m. of four investigated human samples of age-related macular degeneration associated with choroidal neovascularization and four control tissues consisting of choroid and RPE. Level of significance was calculated using DESeq (p = 3.61x10-21). d) Immunohistochemistry for HLA-DR in the RPE/choroid of one control and the CNV membrane of one patient suffering from age-related macular degeneration associated with choroidal neovascularization.
Preferential localization of cells that have undergone recent reoxygenation to the edge of hypoxic areas.Figure 5a) Overview of a tumor induced by deep cortical deposition of approximately 500 Gli36 cells stably transfected with the dUnOHR sensor construct. The perimeter of the developing tumor (white line in the merged picture, lower right panel) was delineated by Hoechst 33342 staining. As observed for dUnaG the reoxygenation sensor was expressed in compact areas with low vascular density. Vascular endothelium was identified by PECAM-1 staining. 30 µm cryosection. Scale bar = 500µm.b) Details of the tumor shown in a). Sparse, distinct cells are characterized by intense green and orange fluorescence (shown as orange) and are located preferentially to the edge of the hypoxic phalanx (white arrows). A number of cells in close proximity of the nearest blood vessel display weak green and orange fluorescence (asterisks), indicative of previously hypoxic environment and subsequent reoxygenation. Scale bar = 50 µm. c) Recently reoxygenated cells (arrows) cluster at the edge of hypoxic areas and are often detected in the proximity of vessels with a homogeneous caliber (double arrows). Scale bar = 100 µm. d) Previously hypoxic cells, frequently undergo apoptosis after reoxygenation. We noted an accumulation of intensely orange fluorescing spots at the edges of compact UnaG positive areas. Scale bar = 50µm. e) TUNEL staining of tumor sections revealed that the tissue areas rich in intense mOrange aggregates colocalized with areas of increased apoptosis. Scale bar = 50µm.