EMBO/sd-character-level-ner huggingface dataset:
Figure 5A. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP in PHSL1-CDC20 ama1∆ spo13∆ control cells and cells containing cdc5-as or ime2-as treated with CMK or 1Na-PP1, respectively, at metaphase I (7 h in SPM). Top, time-lapse series. Bottom, Spc72's presence at SPBs was quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Data information: data are representative of two independent experiments. B. Whole-cell extracts from PHSL1-CDC20 ama1∆ CDC5-ha3 Ha3-SPO13 cells were subjected to α-Ha immunoblotting. Secondary antibodies were detected by near-infrared fluorescence imaging. Top, progression into metaphase I. Bottom, serial two-fold dilutions of the sample collected at t = 10 h in SPM. The Cdc5-ha3/Ha3-Spo13 ratio is ~20. Data information: data are representative of two independent experiments. C. Cdc5 interacts with Ime2. Cdc5-ha3, Ime2, and Spo13 were detected by immunoblotting in whole-cell extracts and α-Ha immunoprecipitates from the indicated strains. Data information: data are representative of two independent experiments. D. Cdc5's polo-box domain (PBD) binds to Ime2. Ha3-tagged versions of the wild-type PBD or the phosphopeptide-binding mutant PBD-FAA were expressed at t = 5.5 h in SPM in cells depleted of Cdc20 and Cdc5. PBD-ha3 and Ime2 were detected by immunoblotting in whole-cell extracts and α-Ha immunoprecipitates. Data information: data are representative of two independent experiments. E. Cdc5-dependent modification of Ime2. PHSL1-CDC20 ama1∆ control cells and cells containing spo13∆ or cdc5-as were treated with CMK (to inhibit Cdc5-as) at t = 8 h in SPM (arrow heads). The panel shows immunoblot analysis of whole-cell extracts. C, sample from proliferating cells. Increased gel mobility of Ndt80 at t ≥ 10 h confirms inhibition of Cdc5-as. The arrow marks the modified form of Ime2. Data information: data are representative of two independent experiments.
a, GFP–LC3-positive dots in tissues of 6-week-old GFP–LC3 mice treated with the indicated peptide (20 mg kg−1 i.f4a, GFP-LC3-positive dots in tissues of 6-week-old GFP-LC3 mice treated with the indicated peptide (20 mg kg−1 i.p., 6 h). A minimum of ten fields was counted per tissue section. Bars represent mean ± s.e.m. for three mice. Similar results were observed in three independent experiments. b, p62 immunoblot of brains of 5-day-old GFP-LC3 mice treated with the indicated peptide (20 mg kg−1 i.p., 6 h). c, Survival curves of 5-day-old C57BL/6J mice infected with CHIKV (106 p.f.u. s.c.) and treated daily with peptide (15 mg kg−1 i.p. beginning 1 day post-infection). d-f, Representative images of WNV envelope antigen and TdT-mediated dUTP nick end labelling (TUNEL) staining (d) (T, Tat alone), quantification of cell death in brain (e) and survival curves (f) for 5-day-oldC57BL/6Jmice infected with WNV (Egypt strain 101, 1 p.f.u. intracerebral (i.c.)) and treated daily with peptide (D-amino acid forms, 20mgkg−1 i.p. beginning 1day post-infection). Images in d are from cerebral cortex day 6 post-infection. Similar results were observed in all regions of the brain for three mice per group. Scale bar, 20μm. Bars in e represent mean±s.e.m. TUNEL-positive cells per unit area of brain for three mice. g d-f, Representative images of WNV envelope antigen and TdT-mediated dUTP nick end labelling (TUNEL) staining (d) (T, Tat alone), quantification of cell death in brain (e), and survival curves (f) for 5-day-oldC57BL/6Jmice infected with WNV (Egypt strain 101, 1 p.f.u. intracerebral (i.c.)) and treated daily with peptide (D-amino acid forms, 20mgkg−1 i.p. beginning 1day post-infection). Images in d are from cerebral cortex day 6 post-infection. Similar results were observed in all regions of the brain for three mice per group. Scale bar, 20μm. Bars in e represent mean±s.e.m. TUNEL-positive cells per unit area of brain for three mice. g g, Geometric mean + s.e.m. viral titres of WNV-infected mousebrains day 6 post-infection. Values represent combined data for 12-20 mice per treatment group from 10 to 12 litters. Data in c and f represent combined survival probabilities for three and four independent litters, respectively, in each group. Similar results were observed in each independent experiment. *P 0.05; **P 0.01; ***P 0.001; NS, not significant; t-test.
Figure 2(A) EdU staining of WT or Mettl14 cKO skin after pulse-chase labeling. Skin samples were counterstained with antibody against β4-integrin. Note reduced EdU label-retaining cells in cKO skin epidermis. Arrows indicate Edu-positive cells. (B) Label-retaining cells in WT or Mettl14 cKO skin were quantified and shown as box plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=19, P<0.01 (Student's t-test). (C) Number of holoclones derived from WT and Mettl14 cKO skin was quantified and shown as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=24, P<0.01 (Student's t-test). (D) Morphology of primary keratinocytes isolated from WT or Mettl14 cKO skin. (E) Expression of Mettl14 and p63 in WT and KO cells was determined by immunoblots with respective antibodies. Immunoblot for GAPDH was used as loading control. (F) Proliferation of WT and Mettl14 null cells in vitro was quantified and shown as dot plots. n=3, P<0.01 (Student's t-test) for Day 6, 9, and 12, and P<0.05 (Student's t-test) for Day 3. Error bar represents S.D. (G-H) CFE (colony formation efficiency) of WT and Mettl14 null cells was determined in vitro. Results were quantified and shown as bar graph (F). n=19, P<0.01 (Student's t-test). Error bar represents S.D. (I) Fluorescence microscopy demonstrates different survival capability of WT and Mettl14 inducible KO cells with or without Tamoxifen (TAM) treatment. (J) Ratio of WT and Mettl14 inducible KO cells in the co-culture model was quantified and shown as dot plots. n=8, P<0.01 (Student's t-test) for KO cells with TAM treatment compared with WT cells or KO cells without TAM stimulation at both Day 7 and 14. Error bar represents S.D.
Interactions of FRH with RNA and ADP.Figure 5(A) 3.8 Å resolution mFo-DFc omit map density (green, calculated with sharpened B-factors and contoured at 2.0) reveals the presence of ADP and a short segment of RNA bound to FRH. (B) 3.8 Å resolution 2mFo-DFc calculated without ADP or RNA contributing to the model. Green electron density shown for ADP and RNA (0.6orange density shown for surrounding protein (0.6. (C) Comparison of Mtr4 and FRHADP binding (FRH in cyan with ADP, RNA, and F179 in orange bonds; Mtr4 in green with ADP, RNA, and F148 in yellow bonds). (c) Unwound DNA from Hel308 (pdb 2PR6; magenta, DNA in orange bonds) overlaid in FRH with RNA and ADP (FRH in cyan with yellow RNA and cyan ADP in stick) after superposition of the respective RecA domains. (d) Hel308 DNA helicase DNA (pdb 2PR6; backbone in orange, bases in gray bonds) after aligning the RecA domains of Hel308 with those of FRH (cyan, RNA in yellow); see also Appendix Fig S4. The superposition indicates that the KOW module is positioned to interact with double stranded RNA that is being unwound by the RecA and DHSCT domains.
Figure 5A. Wild type and KAP114 knockout strains were diluted serially and spotted onto YPD plates in the presence of 1.5 M NaCl. B. The KAP114 knockout strain was transformed with vector control (lane 2) and a plasmid containing wild type KAP114, KAP114 (Δ347-371), or KAP114 (Δ899-956) (lanes 3-5). Wild type and KAP114 knockout strains containing plasmids were serially diluted and grown in minimal medium plates supplemented with 1.5 M NaCl. Growth temperatures are indicated. C. A heatmap generated by three independent biological replicates shows gene expression profiles of wild type and KAP114 knockout strains grown in the presence of 1.5 M NaCl. The Log2 (fold-change) values and the color scale are shown. D. Gene ontology enrichment analysis of up- and down-regulated genes, analyzed separately using Metascape (http://metascape.org). The top 10 significant GO terms (biological pathways and processes) associated with up- and down-regulated genes are shown. The y-axis represents the GO category (ribosomal biogenesis pathways/processes are highlighted in bold), and the x-axis represents the -Log10 (P-value) of GO terms. E, F. (E) qPCR analysis of gene expression in the KAP114 knockout strains (green bars) compared to wild type (black bars) under regular conditions. (F) qPCR analysis of gene expression in the KAP114 knockout strain (green bars) compared to wild type (black bars) grown in high salt conditions. qPCR fold-changes in mRNA expression are relative to control (YPD media, black bars). Actin was used as the internal control. G. ChIP-qPCR assays showing yTBP-GFP binding to the promoter of indicated genes in wild type and KAP114 knockout strains grown in high salt conditions (light green bars) compared to under regular conditions (black bars). Chip-qPCR fold-enrichment is relative to control (YPD media, black bars). H. Endogenous Ran, GFP-yTBP and proliferating cell nuclear antigen (PCNA; loading control) in nuclear fractions isolated from wild type yeast grown in regular and high salt conditions were analyzed by Western blot using antibodies for Ran, GFP and PCNA. I, J. Analysis of Ran and GFP-yTBP in nuclear fractions isolated from wild type yeast grown in regular and high salt conditions. The combined bar and scatter plot shows the band intensity ratios of Ran (I) and GFP-yTBP (J) over PCNA. Each dot represents an individual data point.
Figure 5(A) Fraction of polysomal ribosomes quantified from polysome profiles in the wild type and noNSUN strains subject to a 4 h heat shock at 27°C. ns = non-significant. Data information: In (A , n = 3 biological replicates. In (A), data are presented as mean ± SEM, unpaired two-tailed Student's t-test. (B) Heatmap showing P-site codon occupancy according to the colour scale at 20°C and 27°C. Red and blue refer to enhanced and reduced codon occupancy, respectively, in the noNSUN strain relative to wild type. Leucine and proline codons are marked in red. Data information: In n = 3 biological replicates. (C) Ribosome-protected fragment (RPF) counts in each sample plotted along ife-1 and pat-10 CDS. Vertical grey lines indicate UUG codons. Data information: In , n = 3 biological replicates. (D) Translation efficiency of UUG-enriched, leucine-enriched, proline-enriched and random genes in each sample. A gene was considered enriched in a certain codon when the proportion of this codon in the gene was at least 3-fold higher than the proportion of the same codon across the transcriptome. Data information: In n = 3 biological replicates. In (D), boxplots show the median (central band) and IQR (boxes) ± 1.5 x IQR (whiskers), Welch's t-test, p-value < 0.05.
Bayesian skyline plot (BSP) and phylogenetic tree of human-associated S.Figure 3Bayesian skyline plot showing the population size through time for human-associated S. suis, which is constructed based on a subsample of HAC isolates (n=174). The Y-axis represents the effective population size, and the X-axis is calendar years. The line shows the median estimate of the population size. Blue shading shows 95% highest posterior density. Timed phylogeny of the subsample of HAC isolates (n=174). Maximum clade credibility tree is produced using strict-clock model in BEAST2. Major sub-lineages in Asia are indicated (I, II, III). Acquisition of the 89-kb pathogenicity island is indicated by a red pentagon. Acquisition of the 78-kb pathogenicity island, and the 127-kb MGE are indicated by black and green pentagon, respectively. The blue and red block of heatmaps represent the presence of MGEs and ARGs, respectively. Gray represents absence.
a) GFP-LC3 H1299 cells were treated with 1 μM of the catalytic mTOR inhibitor AZD8055 in fed conditions with DMSO (left) or with 1 μM SAR405 (right).figf5(a) GFP-LC3 H1299 cells were treated with 1 μM of the catalytic mTOR inhibitor AZD8055 in fed conditions with DMSO (left) or with 1 μM SAR405 (right). Cells were fixed, and nuclei were stained using Hoechst 33342 (blue). One representative image of cells out of three independent experiments, acquired using an iCyte imaging cytometer, is shown (X40). White arrows indicate the GFP-LC3 spots, which correspond to the autophagosomes. Scale bars, 10 μm. (b) Histograms show the percentage of positive cells (top) and the GFP intensity (bottom), as measured using an imaging cytometer. Treatment of GFP-LC3 H1299 in fed conditions with 1 μM of AZD8055 and different concentrations of SAR405 is indicated. For GFP intensity, the values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments. (c) The concentration needed to reach 40% inhibition (IC40) of cell proliferation is indicated. The top histograms indicate the IC40 of SAR405 as a single agent and in combination with everolimus in the three cell lines. The bottom histograms indicate the IC40 of everolimus as a single agent and in combination with SAR405. The IC40 values for the combination of the two compounds were determined using rays with an effective fraction ∼0.5, corresponding to compounds that are in equipotent proportion in the mixture (Supplementary Fig. 20). The absolute IC40 determination of both compounds is represented as the geometric mean from two or three independent experiments.
Drug-induced regulation of pyrimidine biosynthesis enzyme DHODH contributes to cell death A Relative mRNA expression levels of DHODH in HCT116 cells treated as indicated for 16h (NEN (N) 1.Figure 5A Relative mRNA expression levels of DHODH in HCT116 cells treated as indicated for 16h (NEN (N) 1.2 µM, Domperidone (Domp, D), Imipramine (Imi), Desipramine (Desi), Amitriptyline (Ami), each 30 µM, Clomipramine (Clomi) 20 µM). Data information: , data are presented as mean (SD) (N=3) and were analyzed by Kruskal-Wallis with Dunn´s post-hoc test (A, left graph), one-way ANOVA with Tukey post-hoc test (A, right graph) In (A), significance is indicated for the comparison of the different treatments to controls. ** p < 0,01; **** p < 0,0001. B Caspase activity in HCT116 cells treated as indicated for 24h (NEN 0.6 µM, Domperidone (Domp) 20 µM, A77126 (A77) 50 µM, JNK inhibitor II (Jnki) 1µM). Data information: data are presented as mean (SD) (N=3) and were analyzed by two-way ANOVA with Tukey post-hoc test C Immunoblot analysis of HCT116 whole cell lysates using antibodies against indicated proteins and treated as follows: NEN and Domperidone for 12h, A77126 for an additional 6h (NEN 0.6 µM, Domperidone (Domp) 20 µM, A771726 50 µM). D Caspase activity in HCT116 cells treated as indicated for 24h (NEN 0.6 µM, Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 20 µM, Clomipramine (Clomi) 10 µM, A77126 (A77) 50 µM). Data information: data are presented as mean (SD) (N=3) and were analyzed by two-way ANOVA with Tukey post-hoc test
Figure 5 ­­­A Western blot analysis showing dynamic expression patterns of FBXW7 and c-MYC during the indicated time points of EB induction. α-TUBULIN was used as a loading control. B Western blot analysis showing FBXW7 and c-MYC expression in control, Mettl5 KO (KO-6B) and KO+HA-MT5WT Day7 EBs, respectively. α-TUBULIN was used as a loading control. C Schematic diagram depicting induction of FBXW7 expression using rtTA-dependent system in Mettl5 KO mESCs. Reverse tetracycline-controlled trans-activators (rtTA) can recognize the TET response element (TRE) and express FBXW7 at the presence of a derivative of tetracycline-doxycycline (Dox). D RT-qPCR analysis of the expression of pluripotency and lineage-specific markers in Day7 FBXW7-induced EBs at the presence or absence of 10 ng/ml Dox. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. *, P < 0.05; **, P < 0.01; ***, P<0.001. E Immunostaining analysis of the expression of pluripotency marker POU5F1, lineage-specific markers FOXA2, T and NESTIN in Day7 FBXW7-induced EBs at the presence or absence of 10 ng/ml Dox. Scale bars represent 100 μm. F Schematic diagram depicting induction of c-MYC expression using rtTA-dependent system in WT mESCs. G RT-qPCR analysis the expression of pluripotency and lineage-specific markers in Day7 c-MYC-induced EBs at the presence or absence of 10 ng/ml Dox. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. *, P < 0.05; **, P < 0.01; ***, P<0.001. H Immunostaining analysis of the expression of pluripotency marker POU5F1, lineage-specific markers FOXA2, T and NESTIN in Day7 c-MYC-induced EBs at the presence or absence of 10 ng/ml Dox. Scale bars represent 100 μm.
Figure 2HEK293, RPE, or U2OS/MycBioID-FAM111A cell lines were induced with doxycycline to express wild-type or mutant FAM111A for 24 h (HEK293), 48 h (RPE), or 72h (U2OS). The colonies were fixed then stained with crystal violet. Cell-cycle profiles of HEK293, RPE and U2OS FAM111A-expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry. C. HEK293 cell lines were induced with doxycycline to express wild-type or mutant FAM111A for 24 h (HEK293) Cell growth was quantitated as total area on ImageJ. Values are mean ± s.e.m. of independent experiments (n = 3). *p<0.05, **p<0.01, ****p<0.0001 (two-tailed unpaired t-test). D. Cell-cycle profiles of HEK293 FAM111A-expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry.
a) Immunoblotting of Atg7-deficient (Atg7F/F:Mx1; Atg7F/F shown here as F/F) and Atg7 Nrf2-deficient (Atg7F/F:Mx1:Nrf2−/−) livers.figf5(a) Immunoblotting of Atg7-deficient (Atg7F/F:Mx1; Atg7F/F shown here as F/F) and Atg7 Nrf2-deficient (Atg7F/F:Mx1:Nrf2−/−) livers. Liver homogenates from mice of the assigned genotypes at 28 days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions. Total, soluble and insoluble fractions were subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies (top section). Total lysates were subjected to SDS-PAGE and analysed by immunoblotting with antibodies against Nqo1, Gstm1 and actin (bottom left section). Nuclear fractions were prepared from the livers of the indicated genotypes at 28 days after injection of poly(I)•poly(C), subjected to SDS-PAGE and analysed by immunoblotting with antibodies against Nrf2 and Lamin B (as control) (bottom right section). Data were obtained from three independent experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11. (b) Immunofluorescence analysis of the cellular localization of p62 and Keap1. Liver sections from mice of the indicated genotypes at 28 days after injection of poly(I)•poly(C) were immunostained with anti-Keap1 (left) and anti-p62 (middle) antibodies. Right: merged images of Keap1 (green) and p62 (red). Each inset in the Atg7-deficient and Atg7 Nrf2-deficient liver panels is a magnified image of the boxed region. Scale bars, 20 μm. (c) Quantitative real-time PCR analyses of Nqo1, Gstm1 and Cyp2a5 in mouselivers. Total RNAs were prepared from livers of the indicated genotypes at 28 days after injection of poly(I)•poly(C). Values were normalized to the amount of mRNA in the Atg7F/F liver. Data are means ± s.d. for three experiments. (d) Liver weight. The weights of the mouse livers of the different genotypes shown at 28 days after injection of poly(I)•poly(C) were measured. Data are means ± s.d. for five mice from each group. Three asterisks, P 0.001 (Student's t-test). (e) Histological analysis of the mouse liver of the indicated genotypes. At 28 days after injection of poly(I)•poly(C), the livers were processed for haematoxylin/eosin staining. Higher-magnification views are shown in the bottom panels. CV, central vein; P, portal vein. Scale bars, 100 μm. (f) Liver function tests of the mice used in d. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were measured. (IU/L, international unit per liter). Data are means ± s.d. for seven mice from each group. Asterisk, P 0.05; three asterisks, P 0.001.
Figure 3(A,B) Spinal cord sections (C1-C4) from 5-month-old mice of indicated genotypes were stained with indicated antibodies (A) and the numbers of NeuN, or CHAT positive cells, as well as IBA1 and GFAP intensities were quantified (B). 3-5 sections/mouse were used for quantification. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001. Scale bar: 20 µm for CHAT staining, 200 µm for other images.
Mitochondrial uncoupling induces targetable metabolic vulnerabilities A HCT116 cells were treated as indicated (Control (Ctrl), 2-DG 100 mM; NEN 1.Figure 1A HCT116 cells were treated as indicated (Control (Ctrl), 2-DG 100 mM; NEN 1.2 µM; FCCP 2,5 µM). Graph shows the ratio of toxicity and viability measured after a 24h treatment. Data information: Data are presented as mean (SD) (N=3) and were analyzed by two-way ANOVA with Tukey post-hoc test Significance is indicated for multiple comparisons as indicated. * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001. B Pie chart depicting numerical proportions of indicated drug categories on total hits from the phenotypic screening using the FDA-approved drug library. C Plot shows results of GPCR screen as ratio of cell toxicity and viability. Domperidone and TCAs highlighted in red and orange, vehicle controls in green and positive toxicity controls in blue. Z′ factor was calculated according to the formula Z′ = 1 - (3(θp +θn)/(μp - μn)), where p is the positive control, n is the negative control, θ is the standard deviation, and μ is the mean. For hit selection a threshold of lower than 3 standard deviations from the median of the negative/ vehicle population was set. D Ratio of toxicity and viability of HCT116, U87 and BxPC3 cells grown in spheroids (3D) and treated as indicated for 24h (BxPC3) or 48h (HCT116, U87) (NEN 1.2 µM; Domperidone (Domp), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM). Data information: Data are presented as mean (SD) (N=3) and were analyzed by two-way ANOVA with Tukey post-hoc test Significance is indicated for multiple comparisons as indicated. * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001. E Percentage of apoptotic cells, determined by measuring Annexin-V and PI positive HCT116 or U87 cells by FACS. The effects were assessed after a 24h or 48h treatment respectively (NEN (N) 1.2 µM; Domperidone (Domp, D) 30 µM, zVAD-FMK (zVAD) 100 µM, Staurosporine 100 µM. Cells were pre-treated for 1h with the zVAD-FMK inhibitor prior to adding the drugs. Data information: Data are presented as mean (SD) (N=3) and were analyzed by one-way ANOVA with Tukey post-hoc test (for (E, upper graph) HCT116 cells) or by Kruskal-Wallis with Dunn´s post-hoc tests (for (E, lower graph) U87 cells). Significance is indicated for multiple comparisons as indicated. * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.