EMBO/sd-character-level-ner huggingface dataset:
Figure 7Schematic diagram showing location of major and minor satellite repeats at pericentric heterochromatin and centric heterochromatin on a mouse chromosome (upper) and organization of mouse major and minor satellite repeats (gray and light gray, respectively). During premeiotic DNA replication and meiotic DNA prophase I, spermatocytes bidirectionally transcribe the long and heterogeneous transcripts encoded by major and minor satellite DNA. The resulting major satellite transcripts associate with nucleosomes and Suv39h, and hybridize with major satellite repeats to stabilize pericentric heterochromatin, which allows faithful kinetochore assembly. Meanwhile, minor satellite transcripts stabilize DNA-binding of CENP-C to complete the centromere identity. To remove excess satellite transcripts, double-stranded satellite transcripts are sliced by Dicer-mediated processing and might be followed by ERI1-mediated siRNA degradation. Meanwhile, single-stranded major satellite transcripts are recognized by MIWI-piRNA complexes and processed to smaller fragments that can effectively form double stranded RNA with their complementary satellite RNA sequence for Dicer degradation. Alternatively, these smaller fragments could be degraded by the 5′-to-3' exoribonuclease XRN1 and the exosome. Consequently, spermatocytes can keep a suitable amount of satellite transcripts for proper kinetochore assembly and normal chromosome segregation during meiotic metaphase I. S, sense; As, antisense; Maj, major satellite; Min, minor satellite; CENP-C, centromere protein c; ERI1, enhanced RNAi-1.
Figure 2A. Ribbon diagram of the yeastMdm12 dimer. The crystal structure of full-length Mdm12 was determined by SAD and refined with native data to 3.1 Å resolution. Lipids bound to Mdm12 are drawn with black stick models. B. Schematic diagram indicating the secondary structure elements and their organization in Mdm12. Three structural elements of Mdm12 are highlighted in different colored boxes. C, D. Surface representations of the Mdm12 dimer are shown in different orientations.
eIF4A is required for complete TORC1 inactivation upon amino acid removal.Figure 1(a-b) eIF4A knockdown in either S2 (a) or Kc167 (b) cells blunts the inactivation of TORC1 upon amino acid removal. Cells were treated with dsRNA targeting GFP as a negative control, or three independent, non-overlapping dsRNAs targeting eIF4A for 5 days and then incubated with complete medium or medium lacking only amino acids for 30 minutes. Representative of 3 biological replicates. (c) Only knockdown of eIF4A, but not of other genes involved in translation, causes elevated TORC1 upon amino acid withdrawal. S2 cells were treated with dsRNAs for 5 days and then incubated with medium containing or lacking amino acids for 30 minutes. After cell lysis, levels of S6K phosphorylation (T398) were measured by dot blot analysis and normalized to total S6K levels. Results are summarized here for all genes involved in translation, and provided in Dataset EV1. (d) Impaired inactivation of TORC1 in response to eIF4A knockdown is most apparent upon partial depletion of amino acids, caused by removal of amino acid subsets. After 5 days of knockdown, S2 cells were treated for 30 min with either complete Schneider's medium (+aa), Schneider's medium lacking all amino acids (-aa) or various subsets of amino acids, as indicated (where EAA "essential amino acids" = H,I,L,K,M,T,W,V). Error bars: std dev. n=3 biological replicates. (e) Time course of amino-acid removal reveals that eIF4A knockdown Kc167 cells maintain elevated S6K phosphorylation up to the maximum possible timepoint of 60 minutes when the cells start dying (see drop in S6K and tubulin levels). Representative of two biological replicates. (f) eIF4A mutant larvae have impaired TORC1 inactivation upon shifting to food lacking amino acids. Control (w1118) or eIF4A1006/1013 1st instar larvae were transferred from standard food to plates containing either standard fly food or PBS/1% agarose+2%sucrose for 1h prior to lysis and immunoblot analysis. Two biological replicates are shown. Representative of 3 biological replicates.
The function of NBS1 in DNA end resection in vivo.Figure 4A schematic representation of the NBS1 knockdown-rescue experiments in U2OS cells. In vivo resection assay using BrdU staining under native conditions to directly detect ssDNA at sites of DSBs. Shown are representative images. In vivo resection assay using RPA2 staining as a marker for ssDNA present at sites of DSBs. Shown are representative images. Quantitation of the experiment shown in (B). At least 100 cells were assessed per condition. The horizontal line represents a median, the box spans from 25th to 75th percentile and the whiskers from 10th to 90th percentile. Quantitation of the experiment shown in (C). At least 100 cells were assessed per condition. The horizontal line represents a median, the box spans from 25th to 75th percentile and the whiskers from 10th to 90th percentile. Quantitation of the single strand annealing (SSA) reporter assay in U2OS cells. All cells were treated with DMSO; where indicated, both PFM03 (50 μM) and PFM39 (50 μM) MRE11 inhibitors were added. Averages shown; n = 4, error bars, SEM. Insert, western blot analysis of NBS1 and CtIP protein levels upon depletion with siRNA. Quantitation of resection activity in NBS1∆N cells complemented with wild type (WT) or NBS1 R28A K160M mutant and either mock depleted (siCtrl) or depleted of CtIP (siCtIP). At least 300 cells were assessed per condition. The horizontal line represents a median, the box spans from 25th to 75th percentile and the whiskers from 10th to 90th percentile. Statistical significance was determined using ordinary one-way ANOVA with Tukey's multiple comparison test, **** (p<0.0001).
atntr1, in contrast to herboxidiene‐treated plants, shows localised decrease in PolII occupancy on alternatively spliced exonsLine charts present ChIP profile of total PolII on examined genes.Figure 4Line charts present ChIP profile of total PolII on examined genes. Black, grey and dashed black lines represent results for Col‐0, atntr1‐1 and herboxidiene‐treated WT plants, respectively. Above each chart, gene structure is shown with black boxes representing constitutive exons; grey boxes, alternative regions; white boxes, promoter region; black lines, introns. Red lines show amplified regions. Above each gene structure, 0.5 kb scale is shown. For each chart, the mean value from three independent experiments is shown. Error bars represent ± SD, **P 0.01 and *P 0.05 of t‐test. Arrows on gene structure show localisation of primers used for splicing analysis by RT‐PCR, which was followed by capillary electrophoresis (results shown in tables next to each gene). Tables represent splicing site selection in mutant and herboxidiene‐treated plants in comparison with wild‐type. Source data are available online for this figure
Figure 1Venn Diagram representing the distribution of proteins identified by both the BioID and the standard AP/MS of REV7, with or without DNA damage (NCS). Selected BioID REV7 results, shown as dot plots. The spectral counts for each indicated prey protein are shown as AvgSpec. Proteins were selected based on and iProphet probability of >0.95, BFDR of <0.05 and ≥10 peptide count. The circle size represents the relative abundance of preys over baits. U2OS EJ5-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).
Nep1r1 and Ctdnep1 downregulation restore NPC levels and suppress INM herniations in dTorKO cells A) mAb414 labeling of 5DO dTorKO fat body cells expressing the dTor cDNA, Luciferase RNAi, or RNAi against Nep1r1, Ctdnep1, or Lipin.Figure 6mAb414 labeling of 5DO dTorKO fat body cells expressing the dTor cDNA, Luciferase RNAi, or RNAi against Nep1r1, Ctdnep1, or Lipin. Nuc, nucleus. The percentage of fat body cells with NE-localized mAb414. ** and **** indicate that Lipin, Ctdnep1, and Nep1r1 RNAi significantly increased the number of dTorKO cells with NE-specific mAb414 labeling compared to the Luciferase RNAi (Luc). Chi-square test followed by individual post hoc Chi-square tests. ** p < 0.01 and **** p < 0.0001. NE ultrastructure in 5DO dTorKO cells. Red arrows, nuclear pores; yellow arrows, abnormal INM herniations. Nuc, nucleus; Cyto, cytosol. The density of NPC detected by TEM in the 5DO dTorKO NE. Each point shows the mean of multiple measurements from an individual nucleus, bars show mean ± SD for the group (n = 5). One-way ANOVA, with Bonferroni's post hoc test. ** p < 0.01, *** p < 0.001 and **** p <0.0001. Same as above except that the density of INM herniations is presented. The percentage of NE in 5DO fat body cells that has normally appearing INM, ONM, and NPC morphology. Points show values from individual nuclei (n = 5). Bars show mean ± SD. Grey zone shows +/- 2x SD of the positive control mean, dotted red line shows mean of dTorKO expressing Luciferase RNAi. One-way ANOVA, with Bonferroni's post hoc test. ** p < 0.01 and **** p <0.0001. The percentage that Lipin, Ctdnep1, and Nep1r1 RNAi normalize the abundance of membrane GL and GPL classes in the dTorKO fat body. 0% "normal" reflects lipid abundance in dTorKO expressing Luciferase RNAi, while 100% "normal" reflects lipid abundance in w- expressing Luciferase RNAi. Percentage normalization was separately calculated for PI, PA, DAG, PC, PE and PS (for dTorKO expressing Lipin, Ctdnep1, and Nep1r1 RNAi). Points show the mean of all lipid class normalization per sample, bars show mean ± SEM for the group.
Figure 8The domestication of the PIF/Harbinger DNA transposon gave rise to two host genes, IDAP1 and IDAP2, which encode a putative DNA transposase and a myb-like DNA-binding protein, respectively. IDAP1 and IDAP2 form a complex in the nucleus where they cooperatively recognize target loci with TE-like structures and recruit the histone acetyltransferase IDM1 to these target loci. Since a previously identified protein MBD7, which recognizes highly methylated regions, also regulates IDM1 targeting, the joint action of MBD7, IDAP1 and IDAP2 may facilitate the targeting of the IDM1 complex to specific loci with multiple epigenetic features. The IDM1 complex then acetylates H3K18 and H3K23 at those loci and facilitates active DNA demethylation mediated by ROS1.
Figure 7Expression of Flag-Fis1-SUMO-2ΔGG, but not Flag-Fis1, in HeLa cells prevents DFP-mediated induction of LC3-II. Flag-Fis1 WT or Flag-Fis1-SUMO-2ΔGG mutant was transfected into HeLa cells for 48 h and the cells were treated with DFP for a further 24 h. Lysate samples were blotted as indicated. Expression of a SUMOylation-deficient Flag-Fis1 K149R mutant in HeLa cells induces LC3-II. Flag-Fis1 WT or Flag-Fis1 K149R mutant was transfected into HeLa cells in which endogenous Fis1 was depleted using siRNA for 48 h. Lysate samples were blotted as indicated. Expressing a SUMOylation-deficient Fis1 mutant reverses the effect of SENP3 knockdown on DFP-induced LC3-II induction. pcDNA3, Flag-Fis1 WT or Flag-Fis1 K149R mutant was transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA (I); siRNA concentration, 20nM), and the cells were treated with DFP for a further 24 h (DFP, 1mM). Lysate samples were blotted as indicated. SENP3 depletion does not abolish mitophagic puncta detected in HeLa cells expressing SUMOylation deficient CFP-Fis1 K149R in the presence of DFP. CFP-Fis1 WT or CFP-Fis1 K149R mutant were transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h, and the cells were treated with DFP for a further 24 h (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Cyan/blue: CFP-Fis1; Magenta, SENP3; Scale bar, 10 μm); Values are presented as mean ± SEM (n=21 cells per condition from two individual experiments; **** p<0.0001; unpaired t-test). Knockdown of SENP3 was further confirmed by immunoblotting (the lower right panel).
Screening the effect of different GPCR-agonists on MEF2-activity.Figure 1 A Neonatal rat ventricular myocytes (NRVMs) were infected with the 3xMEF2-Luc reporter, serum starved for 20 hours and stimulated for 24 hours with different agonists: 100 nM endothelin-1 (ET1), 1 μM sphingosine-1-phosphate (S1P), 10 μM lisophosphatidic acid (LPA), 1 μM WIN55,212-2 (WIN55, cannabinoid receptor agonist), 1 μM isoproterenol (ISO, β-adrenergic receptor agonist), 100 nM angiotensin II (AngII), 10 μM prostaglandin E1 (PGE1), 10 μM prostaglandin E2 (PGE2) or 100 nM prostanoid F receptor agonist fluprostenol (Flupro), 100 nM treprostinil (Trepro, prostacyclin receptor agonist) Data information: values are mean±s.e.m. In (A), the experiment was performed in triplicates, similar results were obtained in 3 different experiments. Student's two-tailed t-test, *,P<0.05 vs. control (ET1, P=0.0013; S1P, P=0.0144; LPA, P=0.026; WIN55, P=0.6205; ISO=0.0399; AngII, P=0.5812; PGE1, P=0.001; PGE2, P<0.0001; Flupro, P=0.0846; Trepro, P=0.1958)  B PGE2 induces the expression of MEF2 target genes. NRVMs were serum-starved for 24 hours, then were stimulated with 1 μM PGE2 for 2 hours and mRNA levels of the indicated genes were determined Data information: values are mean±s.e.m In (B), n=5, technical replicates, Student's two-tailed t-test, *,P<0.05 vs. control (Nur77, P=0.0148; Myomaxin, P=0.0417; Adamts1, P=0.0112; BNP, P=0.0065)  C Correlation between MEF2 activation and NRVM hypertrophy. MEF2 activity was measured as in (A) upon increasing concentration of PGE2. Cell size of NRVMs was determined after 48-hour stimulation with the indicated concentrations of PGE2. Left: representative images of α-actinin stained NRVMs stimulated with DMSO, 300 nM PGE2, or 1 nM ET1. Scale bar is 10 µm Data information: values are mean±s.e.m In (C), NRVM cell size was determined from a minimum of 100 cells of 3 technical replicates upon different concentrations of PGE2, MEF2-activity was parallel assessed from 6 technical replicates. Correlation was statistically analyzed by calculating Pearson correlation coefficient (Pearson r=0.9626, P=0.0021)  D, PGE2 activates MEF2 via EP3 receptor. NRVMs were infected with the 3xMEF2-Luc reporter and serum-starved for 20 hours. The cells were stimulated with DMSO or 1 μM PGE2 for 24 hours in the presence or absence of different EP receptor antagonists: AH6809 (10 μM, EP1- and EP2-antagonist) or 798106 (200 nM, EP3-antagonist) or L161,982 (2 μM, EP4-antagonist) Data information: values are mean±s.e.m In (D), n=3, independent experiments, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, AH6809, P=0.1092; L798106, P=0.0002; L161,982, P=0.5579). The exact n and P values can be also found in the Source Data excel file for Figure 1
Figure 1A. Arginine methylase inhibitor decreases SIRT7 methylation. HEK293T cells expressing Flag-tagged SIRT7 were treated with increasing doses of AdOx as indicated for 24 hrs. After immunoprecipitated with Flag-beads, SIRT7 was analyzed by western blot and detected by an antibody against mono-methylarginine (α-me1). Relative methylation ratio (Ratio) was calculated by normalizing arginine methylation level against the Flag-SIRT7 protein. WCL, whole cell lysate B. R388 of SIRT7 is evolutionarily conserved. Amino acid sequences corresponding to R388 of human SIRT7 were aligned C. PRMT inhibitor reduces arginine methylation of wildtype SIRT7, but not its RK/RF mutants. HEK293T cells expressing wildtype SIRT7 or its RK/RF mutants were treated with or without AdOx for 24 hrs. Arginine methylation of immunopurified SIRT7 was determined by western blot D. SIRT7 is mono- and asymmetrically di-methylated at R388. Flag-tagged SIRT7 and its RK/RF mutants were expressed in HEK293T cells. Immunopurified SIRT7 was blotted with antibodies against mono-methylarginine (α-me1), asymmetric di-methylarginine (α-me2a), and symmetric di-methylarginine (α-me2s), respectively E. R388 site-specific methylation antibody recognizes R388-methylated peptide, but not the unmodified peptide. The unmodified peptide corresponding to R388, R388-monomethylated (R388me1) peptide, R388-asymmetrically di-methylated (R388me2a) peptide were blotted with α-meSIRT7(R388) antibody F. R388-methylated peptide, but not the unmodified peptide, blocks α-meSIRT7(R388) antibody. After incubation with unmodified peptide or R388-methylated peptide (R388me2a), α-meSIRT7(R388) antibody was used to detect immunopurified SIRT7 G. RK and RF mutations disrupt SIRT7 methylation. Flag-tagged wildtype SIRT7 and its RK/RF mutants were immunoprecipitated and blotted with α-meSIRT7(R388) antibody H. SIRT7 is methylated at R388. HEK293T cells ectopically expressing Flag-tagged SIRT7 were treated with increasing concentrations of AdOx for 24 hrs as indicated. SIRT7 protein was pulled down by Flag-beads. Arginine methylation of SIRT7 was detected with a site-specific antibody against R388 methylation [α-meSIRT7(R388)] I-J. AdOx treatment decreases R388 methylation of SIRT7. MEF and L02 cells were treated with AdOx for 24 hrs. Methylation of immunoprecipitated endogenous SIRT7 was determined by western blot (I). The band intensity of western blots was measured to quantify relative R388 methylation ratio (J) (mean ± SD, n=3 experimental replicates, ***p < 0.001, unpaired two-tailed t-test) K-L. Quantification of R388-methylated endogenous SIRT7 in HEK293T and L02 cells. R388-methylated endogenous SIRT7 was captured from HEK293T and L02 cells by using R388 site-specific methylation antibody (K). Input, output, and immunoprecipitate were analyzed by western blot to quantify the percentage of R388-methylated protein (L) (mean±SD, n=3 experimental replicates)
Figure 1(A) CXCL10 assay characteristics. Standard curves of the short, long and total CXCL10Simoa assays are shown. For each curve, limit of detection (LOD) defined as blank+3SD, are shown as horizontal lines. The LOD was 1.7pg/ml for long and short CXCL10 assays and 0.22pg/ml for total CXCL10. Standard curves were fitted using the 4 Parameter Logistic non-linear regression model. Samples reporting a signal below the LOD were replaced with 1pg/ml for short and long CXCL10. Plasma from healthy individuals receiving (B) placebo (n=9) or (C) sitagliptin (n=27) were analyzed by Simoa. Blood samples were collected at screening visit (SV) and day 0 (D0) before treatment, at day 3 (D3), day 14 (W2) and day 28 (W4) under treatment and 5 weeks after treatment interruption (W9). (C) Plasma from chronic HCVpatients receiving sitagliptin (n=3) were collected before (D0) and weekly during sitagliptin treatment (W1 W3). Antagonist CXCL103-77 (short CXCL10, in blue), agonist CXCL101-77 (long CXCL10, in red) and total CXCL10 (in black) levels are shown. Each dot represents a donor and bars are at the median. Grey areas highlight the period under placebo or sitagliptin treatment. Statistical analysis of (B) and (C) was performed using non parametric Friedman's test, ns: non significant, **: P0.01, ****: P0.001. For (C), additional size effect analysis was performed and Cohen's d values are reported. No statistical analysis was performed in (D) due to sample size, nd: non-determined.
a) We transfected MN-1 cells with the 4X-CLEAR luciferase reporter and with BFP-tagged TFEB or BFP empty vector, and then measured luciferase activity.figf5(a) We transfected MN-1 cells with the 4X-CLEAR luciferase reporter and with BFP-tagged TFEB or BFP empty vector, and then measured luciferase activity. Marked induction of luciferase activity occurred with BFP-TFEB; n = 3 independent experiments, ***P 0.001, t-test, WT t(4) = 48.79; ***P 0.001, t-test, AR 24Q t(4) = 22.91; ***P 0.001, t-test, AR 65Q t(4) = 14.02. Results for each line were normalized to the BFP-empty luciferase activity. (b) We transfected MN-1 AR65Q cells with the 4X-CLEAR luciferase reporter and with BFP-TFEB or BFP empty vector and subjected the MN-1 AR65Q cells to conditions that promote TFEB activation, as shown. MN-1 WT cells were also transfected with the 4X-CLEAR luciferase reporter and BFP empty vector and subjected to the identical treatments for control purposes. Results are shown normalized to untreated MN-1 WT cells expressing BFP-empty. Untreated, F = 182.7; starvation, F = 63.64; NH4Cl, F = 291.9; Rapamycin, F = 3128; ANOVA with post hoc Tukey test. **P 0.01, ***P 0.001. (c) We transfected MN-1 AR65Q cells with the mCherry-EGFP-LC3 construct and with either BFP-empty or BFP-TFEB. Autophagic vesicles forming in BFP-expressing cells were then detected as red, green or yellow puncta, as shown. Scale bar, 20 μm. (d) Left, quantification of autophagic vesicle type. Note marked reduction in autophagosomes in MN-1 AR65Q cells expressing BFP-TFEB; n = 3 independent experiments, F = 16.596, ANOVA with post hoc Tukey test. **P 0.01, ***P 0.001. Right, calculation of the autophagy index; n = 3 independent experiments, F = 24.43, ANOVA with post hoc Tukey test. **P 0.01. Data are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.
Screening of target miRNAs silencing ESC self-renewal.Figure 1Screening strategy. miRNAs potentially targeting important ESC pluripotency factors meanwhile enriched in MEFs or up-regulated during EB differentiation were selected as candidate miRNAs. Candidate miRNAs silencing ESC self-renewal were identified by the function analyses in Dgcr8-/- ESCs. Distribution of candidate miRNAs. There were 44 miRNAs enriched in MEFs (red region), 53 miRNAs up-regulated during EB differentiation (blue region) and 454 miRNAs with binding sites to the 3′ UTR of pluripotency factors (yellow region). The gray region represents 52 selected candidate miRNAs in silencing self-renewal. List of candidate miRNAs. Left panel showed candidate miRNAs enriched in MEFs comparing with ESCs. Right panel showed candidate miRNAs up-regulated during EB differentiation. Gray highlighted the known miRNAs suppressing ESC self-renewal. Red marked the same miRNAs existed in both of the two panels. XY and XX indicated ESCs derived from male or female mouse respectively. NA meant D5/D0 was extremely high as the expression value in D0 was zero. Colony formation assay of miRNA mimic transfected-Dgcr8-/- ESCs. There were 17 miRNAs that decreased the colony forming ability, 15 miRNAs that did not affect colony formation and 8 miRNAs enhanced colony formation of ESCs. Representative pictures are shown. Full data are shown in Supplementary Fig S1.