EMBO/sd-character-level-ner huggingface dataset:
Figure 1IncuCyte cell proliferation curves of PC3 cells treated with the indicated concentrations of ASNase. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001. IncuCyte cell proliferation curves for ASNS knockout (sgASNS) and control (sgNon-targeting) PC3 cells in the absence and presence of ASNase. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001. Flow chart for a genome-wide CRISPR-Cas9 functional screen in PC3 cells. Volcano plots for the MAGeCK pipeline analysis of the sgRNA abundance from the screen. Green dots indicate positive controls and red dots indicate candidates with a fold discovery rate (FDR) <0.003. IncuCyte cell proliferation curves of SLC1A3 knockout (sgSLC1A3) and control (sgNon-targeting) PC3 cells in the absence and presence of ASNase treatment. #3 and #4 represent 2 different sgRNAs targeting SLC1A3. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001. Radioactive labeled aspartate and glutamate uptake measurement in control (sgNon-targeting) and SLC1A3 knockout (sgSLC1A3) PC3 cells. #3 and #4 represent two different sgRNAs targeting SLC1A3. Radioactive labeled leucine uptake was used as a control. Data was normalized to the reads of control PC3 cells. Endogenous levels of aspartate, asparagine, glutamate and glutamine in control (sgNon-targeting) and SLC1A3 knockout (sgSLC1A3) PC3 cells with or without ASNase for 3 days. Median peak intensity was used for the read normalization. IncuCyte cell proliferation curves of SLC1A3 knockout (sgSLC1A3#3) PC3 cells treated with ASNase and supplemented with either esterified-aspartate (6mM) or esterified-glutamate (6mM), and esterified-leucine (6mM) as a control. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001.
Fig.Figure 4The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. General cell survival (calcein-AM)/death (EthD-1). (B) Cleaved caspase-3+ apoptotic cell death. (C) Cellular senescence (β-gal staining). (D) DNA damage estimated by γH2AX staining. Cells with >4 γH2AX foci in the nucleus were counted. (E) Mitochondrial ROS (MitoSox). (F) Mitochondrial membrane potential (MitoID). The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. Seahorse test. Terminal differentiation of neural stem cells (NSCs) was induced and the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analyzed on the NSCs at differentiation day 4. p<0.001*, n=7 (WT), 10 (mutant), t-test. The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. Western blot analyses for ER stress marker proteins. The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. Resistance of mDA neurons against mitochondrial toxins. Differentiated mDA neurons were exposed to the toxins indicated for 1 h, and viable TH+ mDA neurons were counted. Data are expressed as % TH+ cells relative to untreated. Three cultures were analyzed for each cell line. N=3 independent experiments, *Significance at p<0.05. Scale bars, 100 µm.
a) Cytokine secretion by LPS-primed Map1lc3b+/+ and Map1lc3b−/− (a) or Becn1+/+ and Becn1+/− (b) peritoneal macrophages incubated for 15 min with glybenclamide (Glyb; 100 μM (a) or 50–100 μM (b)), Bay 11-7082 (Bay; 12 μM) or DMSO (–), followed by ATP.figf6(a) Cytokine secretion by LPS-primed Map1lc3b+/+ and Map1lc3b−/− (a) or Becn1+/+ and Becn1+/− (b) peritoneal macrophages incubated for 15 min with glybenclamide (Glyb; 100 μM (a) or 50-100 μM (b)), Bay 11-7082 (Bay; 12 μM) or DMSO (-), followed by ATP. (c) Cytokine secretion by LPS-primed wild-type (WT) and NALP3-deficient (NALP3-KO) macrophages incubated with rotenone, followed by stimulation for 1 h with ATP. (d) Quantitative PCR analysis of cytosolic mtDNA in wild-type and NALP3-deficient BMDMs left unstimulated (-) or stimulated with LPS, followed by treatment for 15 or 30 min with ATP (left), and immunoblot analysis of cytosolic cytochrome c and activation of caspase-1 in the cells at left (right). (e) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed wild-type, NALP3-deficient or ASC-deficient (ASC-KO) BMDMs stimulated for 15 min with ATP. *P 0.05, NALP3-deficient or ASC-deficient versus wild-type treated with LPS and ATP (Student's t-test). (f) Flow cytometry of untreated macrophages (control) or of LPS-primed macrophages left unstained or stained with MitoSOX, then stimulated for 15 min with ATP. (g) Flow cytometry of untreated macrophages (control) or of macrophages stimulated with LPS and ATP, stained with MitoTracker Deep Red. Numbers above bracketed lines indicate percent cells with loss of mitochondrial membrane potential. (h) Flow cytometry of LPS-primed wild-type, ASC-deficient and NALP3-deficient peritoneal macrophages stimulated for 30 min with ATP and stained with annexin V. Data represent three experiments (mean and s.d. in a-e).
Figure 6A-C. FACS analysis of constitutively expressed sfGFP fusions to chuA (A), cirA (B), or bfr (C) in the presence of Esr41 (orange), RyhB (blue), or a scrambled RNA control (red). The histogram shows GFP fluorescence for each sRNA-mRNA fusion. D. Esr41 confers resistance to Colicin 1A and 1B in the sensitive background, E. coli DH5alpha. Top agar lawns of DH5alpha expressing a control scrambled RNA (pJV300), Esr41 (pZE12::esr41), or RyhB (pZE12::ryhB) were spotted with colicins indicated (top). Zones of clearing indicate sensitivity to the tested colicin. E. Esr41 confers a competitive disadvantage on EHEC under iron-limiting conditions. Wild type EHEC and isogenic ∆esr41 strain (black), or EHEC ∆esr41and the chromosomally repaired strain EHEC ∆esr41::esr41 were inoculated at equal densities and cultured in MEM-HEPES media for 3 days. The proportion of each strain was determined at each time point (days) and is expressed as a ratio relative to the starting inoculum where 1 is an equal fitness (see Appended Supplementary Methods).
Figure 5Munc18-1 knockout neurons expressing G544D Munc18-1 were plated on a microelectrode array before addition of compounds (0 h) or 48 hours after vehicle (DMSO) or compound addition. Munc18-1 knockout neurons expressing G544D Munc18-1 were subjected to analysis of mean firing rate before addition of compounds (0 h) or 48 hours after vehicle (DMSO) or compound addition. 16 electrodes per well were analyzed for neuronal firing (A; purple boxes indicate network activity). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA and Dunnett's multiple comparison test, or Kruskal-Wallis test followed by Dunn's post-hoc test; n = 11-16 independent experiments; exact n and p values are shown in Appendix Table S1).
Fig.Figure 1(A) Yeast MRPs having uncleaved N-termini that can be tracked in the mitoribosome structure (PDB:5MRC) score much lower with MTS prediction algorithms (average of TargetP2 and MitoFates) compared to other MRPs that have their N-termini cleaved off or are not present in the structure (so might be flexible and outside the mitoribosome body). Mean ± SD is indicated by the bars, no replicates were performed as the values are predictions. See Fig. EV1 for more detailed data. (B) Schematic of MRP truncations used to characterize targeting properties of MRP N-termini: MRPFull as control, MRP∆30 to check if the N-terminus is necessary, MRP1-30 to check if it is sufficient (top) and the schematics of expected GFP localization in case the N-terminus is MTS-like (necessary and sufficient) or not (bottom); Micrographs collected in the GFP channel for each truncation (columns) of each studied MRP (rows) grouped by the N-terminus targeting properties based on theoretical expectation summarized in (B) with the MRPs possessing MTS-like N-termini in panel (C) Micrographs collected in the GFP channel for each truncation (columns) of each studied MRP (rows) grouped by the N-terminus targeting properties based on theoretical expectation summarized in (B) MRPs with intermediate phenotype in panel (D) and MRPs without N-terminal signal in panel (E), for each MRP a yeast gene name and new nomenclature protein name is shown on the left. Scale bar for all micrographs is 5 µm.
Figure 3A. Volcano plot of differentially expressed genes obtained by DESeq2 analysis in Mettl3 or Mettl14 null tumors compared to control tumors. Significantly upregulated or downregulated genes are plotted in red and blue points, respectively. n.s, non-significant. B. Venn diagrams showing 202 significantly co-upregulated genes and 28 significantly co-downregulated genes in the indicated tumors. C. Meta-enrichment analysis summary for 202 significantly co-upregulated genes as indicated in (C). D. Consensus m6A motifs and P value identified by HOMER from two biological replicates, Student's t-tests. E. Schematic workflow for analysis of Mettl3 and Mettl14 downstream genes and identified genes or peaks number. F. Representative genes with m6A sites generated by integrative genomics viewer. Data is representative of duplicates with similar results. Red represents reads coverage of IP sample and blue represents reads coverage of input sample. Rectangular cyan shade represents the m6A peaks located on transcripts. G. m6A enrichment of Stat1 and Irf1 was examined by m6A RIP-qPCR in control, Mettl3, or Mettl14 depleted CT26 tumors as indicated. Ctla4 functioned as a m6A negative control (Wang et al., 2019) Data are mean ± s.d. * * P < 0.01 by Student's t-tests. H. Immunoblots of p-Stat1 (phosphorylated), Stat1 and Irf1 were carried out in the indicated tumors in triplicate with Gapdh as a loading control. I. Tumor growth from CT26 cells with Mettl3, Mettl14, Mettl3/Stat1, Mettl3/Irf1, Mettl14/Stat1 or Mettl14/Irf1-depleted genes and control under treatment of PD-1 antibody as indicated. n, the numbers of mice. Data are mean ± s.e.m of the indicated number of mice in each group.
Figure 4Confocal image of pmiR396a::H2B:GFP. Normalized read counts from AGO1-IP-Seq and independent northern analysis of AGO1-loaded miR396. Representative confocal image of the pGRF2::H2B:GFP translational fusion. Focused miRoot analysis of the GRF2 translatome signal in WT versus hyl1 columella. Graph: GRF2 normalized read counts. Representative confocal images of the pGRF2::GRF2:GFP translational fusion in WT or hyl1 backgrounds. Focused miRoot analysis of the PLT1 translatome signal in WT versus hyl1 columella. Schematic demonstrating multiple principles of miR396-mediated transcript regulation.
Figure 7Schematic diagram of the ANG prevention model. Mice (n = 8) were pre-treated with 1.25 mg/kg recombinant ANG protein and then subjected to DSS induction. Western blotting of ANG in colonic tissue from WT mice (n = 2) at indicated time point in the ANG prevention model. The grayscale of ANG band in WT mice with no recombinant ANG and DSS treatment (untreated) is arbitrarily set as 1. Body weight loss of the pre-treated WT colitis mice (n = 8). DAI of the pre-treated WT colitis mice (n = 8). Colon length of colon section from the pre-treated WT colitis mice on day 8 (n = 8). serum FITC-dextran level of colon section from the pre-treated WT colitis mice on day 8 (n = 8). representative HE staining image, and (H) histopathological score of colon section from the pre-treated WT colitis mice on day 8 (n = 8). Schematic diagram of the ANG treatment model. Mice (n = 8) were treated with DSS for 7 days, and simultaneously administrated with 1.25 mg/kg recombinant ANG during day 4-6. Western blotting of ANG in colonic tissue from WT (n = 2) mice at indicated time point in the ANG treatment model. The grayscale of ANG band in WT mice with no recombinant ANG and DSS treatment (untreated) is arbitrarily set as 1. Body weight loss of the treated WT colitis mice (n = 8). DAI of the treated WT colitis mice (n = 8). Colon length of colon sections from the treated WT colitis mice on day 8 (n = 8). serum FITC-dextran level of colon sections from the treated WT colitis mice on day 8 (n = 8). representative HE staining image, and (P) histopathological score of colon sections from the treated WT colitis mice on day 8 (n = 8).
Cytoplasmic IMC extensions in concavin(-) sporozoites.Figure 4A. Transmission electron micrographs of wild type sporozoites with highlighted secretory organelles in magenta, IMC in yellow, microtubules in green and plasma membrane in blue. Scale bar 500 nm. B. Transmission electron microscopy of concavin(-) sporozoites with highlighted secretory organelles as in A; rhoptries in red and nucleus in turquoise. Scale bars: 500 nm. C. Reconstruction of SEM serial sections of a complete rounded up concavin(-) sporozoite; plasma membrane in blue and IMC in yellow. Scale bar 1µm. In total 6 parasites were reconstructed from two different preparations.
Fig.Figure 3(A) Immunostaining for the centrosome marker γ-tubulin (TUBG) on Ctrl and Sas-4−/− primary mESCs. The bottom panels are magnifications of the areas marked in the top panels. Scale bars = 20 μm (top) and 10 μm (bottom). (B) Immunostaining and quantification for p53 on Ctrl and Sas-4−/− primary mESCs in pluripotent and partially differentiated (diff.) conditions. The quantification of p53 fluorescence intensities was normalized to Ctrl (Four independent experiments). ** p < 0.01, * p < 0.05 (two-tailed student's T-test, the comparison of the genotypes in the "Pluripotent" condition was not significant using the one-way ANOVA with Tukey's multiple comparisons tests). Bars represent mean ± s.d. Scale bar = 50 μm. Pluripotent Ctrl: 1.00 ± 0.02 (n=2030 cells); pluripotent Sas-4-/-: 1.17 ± 0.10 (n=2170); partially diff. Ctrl: 0.62 ± 0.13 (n=2745); partially diff. Sas-4-/-: 1.12 ± 0.24 (n=2000). (C, D) Three-day growth curves of WT and Sas-4−/− (C) or p53−/− and Sas-4−/− p53−/− (D) primary mESCs in the indicated conditions starting with 105 cells on Day 0 (Four independent experiments). ** p < 0.01, * p < 0.05 (two-tailed student's T-test). Bars represent mean ± s.d. For details of the measurements, see Materials and Methods (Tables 3 and 4).
Figure 1A. Confocal images show Iba1-immunoreactive microglia and macrophages in RPE/choroid whole mount preparations 48 hours after laser coagulation and intravitreal application of low (0.2 µg) and high (3 µg) dose polySia avDP20 or PBS-vehicle. Laser spots of vehicle-injected SIGLEC11 transgenic (tg) and wild type control animals revealed strong accumulation of activated microglia and macrophages, which was effectively decreased in polySia avDP20-injected SIGLEC11 tg animals and to a lower extent in wild type controls. Representative images out of at least three independent experiments are shown. Scale bar: 50µm.B. Quantification of the average pixel intensity of Iba1-positive area in laser spots reflects accumulation of reactive microglia/macrophages on RPE/choroid whole mounts. In comparison to the accumulation of reactive microglia/macrophages in control animals, there is a reduced pixel intensity found in polySia avDP20-treated SIGLEC11 tg animals. Notably, treatment with a high polySia avDP20 dose (3 µg) also reduced accumulation of microglia/macrophages in laser spots of wild type animals. Data show mean +/- SD. * p< 0.05, ** p< 0.01, *** p< 0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 24 spots), WT 0.2 µg Sia (n = 37 spots), WT 3 µg Sia (n = 20 spots), SIGLEC11 tg PBS (n = 25 spots), SIGLEC11 tg 0.2 µg Sia (n = 35 spots), SIGLEC11 tg 3 µg Sia (n = 31 spots), WT PBS vs WT 3 µg Sia p = 0.0147, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p = 0.0046, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p = 0.0071, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p < 0.0001. C. Confocal images of retinal whole mounts show Iba1-immunoreactive microglial cells. Retinal microglia of polySia avDP20-treated SIGLEC11 tg mice had a more ramified microglial morphology in the laser spot compared to PBS-vehicle treated mice. Interestingly, high polySia avDP20 dose (3 µg) also exerted a weak therapeutic effect in wild type animals. Representative images out of at least three independent experiments are shown. Scale bar: 20 µm.D. Percentage of retina showing activated microglial cells within the laser spots was quantified. PolySia avDP20 reduced the percentage of laser spots with activated microglia in SIGLEC11 tg animals and at high dose (3 µg) also in wild type controls. Data show mean +/- SEM. * p< 0.05, ** p< 0.01, ***p<0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 6 retinas), WT 0.2 µg Sia (n = 8 retinas), WT 3 µg Sia (n = 6 retinas), SIGLEC11 tg PBS (n = 8 retinas), SIGLEC11 tg 0.2 µg Sia (n = 7 retinas), SIGLEC11 tg 3 µg Sia (n = 6 retinas),WT PBS vs WT 3 µg Sia p = 0.0052, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p = 0.0005, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p = 0.0159, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0.001.
Figure 4A HeLa cells were transfected with USP19 siRNA or scrambled siRNA and protein extracts were harvested after MG132 (10 μM) treatment for 3 h. Protein extracts were immunoprecipitated using anti-Beclin-1 antibody or with IgG as a negative control and analyzed by immunoblot using anti-ubiquitin and anti-Beclin-1 antibodies. B Lysates of 293T cells transfected with plasmid expressing Flag-Beclin-1 and HA-tagged ubiquitin (HA-Ub (wild type), K6-linked-Ub, K11-linked-Ub, K27-linked-Ub, K29-linked-Ub, K33-linked-Ub, K48-linked-Ub or K63-linked-Ub), together with the empty vector or expression vector of Myc-USP19 and treated with MG132 (10 μM) for 3 h were immunoprecipitated with anti-Flag and immunoblotted with anti-HA. C HeLa cells were transfected with USP19-specific siRNA and plasmid encoding Flag-Beclin-1, plus K11-linked-Ub. Protein was harvested after MG132 (10 μM) treatment for 3 h and immunoprecipitated with anti-Flag and immunoblotted with anti-HA. D 293T cells were transfected with plasmid for Flag-Beclin-1 and HA-K11-Ub, together with the empty vector or expression vector for Myc-USP19 (WT, CS or CS/HA) and treated with MG132 (10 μM). Cell lysates were subjected to immunoprecipitation with anti-Flag and immunoblot analysis with anti-HA. E Purified ubiquitinated Beclin-1 was incubated with immunopurified Flag-USP19 in vitro in deubiquitinating buffer. The immunoblot was probed with anti-HA.
CatSper inhibitors abolish chemoattractant‐ and cGMP‐induced Ca2+ influxCa2+ signals in sperm evoked by photorelease (at t = 0) of resact from caged resact in the presence of the CatSper inhibitor MDL12330A.Figure 5A Ca2+ signals in sperm evoked by photorelease (at t = 0) of resact from caged resact in the presence of the CatSper inhibitor MDL12330A. B Ca2+ signals evoked by intracellular photorelease (at t = 0) of cGMP in sperm loaded with caged cGMP in the presence of the CatSper inhibitor MDL12330A. C Normalized dose-response relation for inhibition of the resact‐ and cGMP‐induced Ca2+ signals shown in (A, B) (Ki = 6.2 and 4.3 μM, respectively). D Normalized dose‐response relation for inhibition of the resact‐ and cGMP‐induced Ca2+ signals shown in Supplementary Fig S4 by the CatSper inhibitor mibefradil (Ki = 7.7 and 20.9 μM, respectively).
x stabilizes Bim protein levels in the ERK-degradation pathway in 293FT cells, 1205Lu melanoma and HCC827 NSCLC cells.Figure 6A 293FT-TetR-3xFlagUsp27x cells were treated with PMA [16.2 nM] plus QVD [10 µM] and 3xFlag-Usp27x was induced by dox at the same time for 24h followed by addition of cycloheximide (CHX). Cells were harvested at the indicated time points, and levels of BimEL were determined by Western blotting. Similar results were obtained in n=3 separate experiments. Cut membranes shown were from one membrane with same exposure time. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point. B 1205Lu melanoma cells carrying dox-inducible GFP-Usp27x were treated with dox for 24h. Cycloheximide (CHX, 1µg/ml) was then added. Cells were harvested at the indicated time points, and levels of BimEL were determined by Western blotting. GFP-Usp27x was detected with anti-GFP-antibodies. Similar results were obtained in n=3 separate experiments. A second experiment is shown in the right panel also including 1205Lu melanoma carrying dox-inducible GFP-Usp27xC87A mutant. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point set to 100%. The starting point for the non-Usp27x/C87A inducing cells (-dox) is shown in lane 1 for experiment one and two (-dox, - CHX). Cut membranes shown were from one membrane with same exposure time. Same results were obtained for WM1158 melanoma cells (data not shown). C HCC827 cells were induced to express GFP-Usp27x with dox as indicated. 24h later cycloheximide (CHX, 1µg/ml) was added for the indicated time. BimEL was detected after the indicated times of treatment by Western blotting. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point. Similar results were obtained in n=2 separate experiments. Cut membranes shown were from one membrane with same exposure time.
DNA methylation analysis of stem cells at different developmental stages.Figure 6(a) CG, CHG, and CHH methylation at chromosome 3 in stem and non-stem cells. The inset for CHG methylation shows a magnification of the pericentromeric region. (b) Metaplots of DNA methylation at CG, CHG, and CHH sites for genes (top) and TEs (bottom). + = stem cells; - = non-stem cells, D7/14/35 = nuclei from 7/14/35 day-old plants, S7/14 = nuclei from 7/14 d-old above-ground seedlings.