EMBO/sd-character-level-ner huggingface dataset:
5-AzadC + Panobinostat and 5-AzadC + Romidepsin treatments do not induce global T-cell activation.Figure 9A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented. A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented. A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented. A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.
Fig.Figure 2A) HeLa cells transfected with indicated plasmids expressing mouse (m) or human (h) DGKk were analyzed by immunofluorescence confocal microscopy for HA tag (red) and Dapi staining (blue). ∆C1-hDGKk construct bears deletion of phorbol ester C1 domain (328-449). B) Immunoblots and quantification of lysates from Cos-1 cells transfected with plasmid pCI-HA-∆N-DGKk, untransfected (NT), or plasmid pCI control, with the indicated amount of plasmid (µg) and after 24h, incubated with puromycin (20µg/ml, 30 min) to measure rates of protein synthesis. GAPDH was used as a loading control. 0 indicates no puromycin treatment, -/+ indicates treatment without or with DGK inhibitor (DGKi) 3 µM R59022 and 0.2 µM R59949 at 6 µM, 15 min. Densitogram of puromycin incorporation is presented as change relative to mock transfected conditions. C) Representative immunofluorescence staining of cortical neuron cultures transduced at 8 DIV (days in vitro) with 10e9 VG/ml of AAVRh10-GFP or AAVRh10-∆N-DGKk and assessed after 5 days using anti-MAP2 and anti-HA for ∆N-DGKk or direct 488 nm excitation for GFP. DAPI was used to visualize nuclei on merged images. D) Representative immunoblots of lysates from WT and Fmr1-KO cortical neurons transduced with AAVRh10-∆N-DGKk (AAV ∆N-DGKk), at the indicated titers (VG/ml culture volume) and quantification of phosphorylation and total levels of eIF4E. GAPDH was used as a loading control. For quantification, the phospho-protein signal was normalized first against total protein signal and is presented relative to the signal for WT culture.
Figure 6(A) Pairs of loci were labeled with YFP-ParBpMT1/parSpMT1 and mCherry-ParBP1/parSP1 at genomic positions 400 kb and 600 kb (inter-domain) or at 600 kb and 800 kb (intra-domain). Box plots show the distribution of inter-focus distances for cells of different sizes. Asterisks indicate statistical significance (***, Student's t-test p-value <0.001; ns, not significant). For the region of the chromosome containing the labeled loci (indicated by dashed grey lines), the corresponding region of the Hi-C map generated at each time point indicated is shown, with the same region from wild-type cells shown at the bottom. For complete Hi-C maps, see Fig EV4; for representative microscopy images, see Fig S3. (B) Same as panel A but for a strain producing CtrA(D51E) Δ3Ω. (C-D) Same as panels A-B, but with pairs of loci labeled with YFP-ParBpMT1/parSpMT1 and mCherry-ParBP1/parSP1 at genomic positions 1400 and 1600 kb (inter-domain) or at 1600 and 1800 kb (intra-domain). Note that differences in Hi-C chromosomal domain sharpness reflect differences in interaction patterns as the color scale and image contrast settings are identical across each time series.
Figure 7Amino acids promote the translocation of mTORC1 to lysosomes. On lysosomal surface, mTORC1 can be activated by the small GTPase Rheb downstream of the growth factor signaling. In this study we discovered that ArfGAP1 interacts mTORC1, likely on some unknown membrane vesicles through its ALPS motifs (not shown here), and inhibits the lysosomal translocation and activation of mTORC1.
Figure 2(A) U2OS WT or PIF1-KO cells were treated with the indicated concentrations of HU (left) or APH (right) for 72 hr, and the cell viability assay was performed. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ***P < 0.001. (B) U2OS WT or PIF1-KO cells were labeled with CldU for 30 min followed by incubation with 2 mM HU for 2 hr and then IdU for another 30 min. Labeled cells were processed for DNA fiber analysis. Representative images of stalled or restarted forks and forks with new origin firing were shown (left). The percentage of restarted forks was quantified by analyzing of 110-130 fibers for each experiment (right). Experiments were repeated four times for each sample. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ***P < 0.001. (C) Schematic drawing of the EGFP-BIR-5085 reporter and the repair steps leading to the repair product expressing EGFP after Cas9n/sgRNA-5085 expression. (D) U2OS (EGFP-BIR-5085) cell lines carrying Dox-inducible Cas9/sgRNA-5085 (Dox-Cas9) or Cas9n/sgRNA-5085 (Dox-Cas9n) were incubated with or without Dox (5 µg/ml) and assayed by FACS analysis 2 days later (left). U2OS (EGFP-BIR-5085, Dox-Cas9 or Dox-Cas9n) cells expressing shRNAs RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were incubated with 5 µg/ml Dox, and FACS analysis was performed after 2 days (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ***P < 0.001. (E) Track length of single EGFP-positive clones derived from U2OS (EGFP-BIR-5085) cells after Cas9/sgRNA-5085 (n=47) or Cas9n/sgRNA-5085 (n=39) expression was analyzed by sequencing of the PCR products from genomic DNA covering the repair junctions.
Figure 3Representation of the two Mettl5 mutant alleles generated in this work, consisting either of a two amino acid deletion upstream of the methyltransferase domain (top, Mettl5∆2AA), or of a frameshift mutation leading to a premature stop codon (bottom, Mettl5fs). LC-MS/MS measurements of m6A levels in total RNA of WT and Mettl5 mutant flies. Bars represent mean ± standard deviation of measurements of three biological replicates. ***P<0.001 (two-tailed t-test). Purified 18S rRNA analyzed for its m6A content by quantitative HPLC. The 18S rRNA was extracted from 40S subunits isolated on sucrose gradients. The calibration control is a commercial source of m6A (in grey). m6A elutes at 48 min. Pre-rRNA processing in Drosophila: four mature rRNAs (the small ribosomal subunit 18S, and the large ribosomal subunit 5.8S, 2S, and 28Sa and 28Sb) are produced by sequential RNA cleavage following two alternative pathways, as depicted. Processing sites are indicated (1 to 6). The major pre-rRNA intermediates (a, b, c, and d) are highlighted. Mature rRNA analysis on ethidium-stained denaturing agarose gels. The same amounts of total RNA extracted from the indicated flies, and from S2R+ cells depleted or not of Mettl5, were loaded. The 28S/18S ratio was established by densitometry. Analysis of 18S rRNA maturation in WT and mutant flies (Mettl5fs). Total RNA extracted from the indicated animals was resolved on denaturing agarose gels and processed for northern blotting with specific probes (complementary to ITS1 or ITS2 sequences). The pre-rRNAs detected accumulate to normal levels indicating that processing is unaffected.
Figure 4(A) Schematic representation of the experimental design. The hybrid TX1072 mouse female ESC line was used, in which Xist can be induced from the endogenous B6 allele. Time course nChiP-seq for H4K20me1 was performed and compared to a published H3K27me3 dataset (GSE116480)(Zylicz et al., 2019). (B) Quantification of average H4K20me1 (blue) and H3K27me3 (red) enrichment at the Xi (B6 allele) compared with t = 0 h in 10-kb windows spanning the whole chromosome. Shading is the interquartile range. (C) As in (B) but shown is normalised B6 enrichment to the average accumulation at 24 hrs. (D) Pairwise comparison of H4K20me1 and H3K27me3 accumulation dynamics (ED50) at the X chromosome. All 10-kb windows with ED50 < 24 h are plotted. * aired Wilcoxon rank sum test P-value <0.05. (E) H4K20me1 (blue) and H3K27me3 (red) accumulation across the Xi after 12 h of DOX treatment. The black line is a locally estimated scatterplot smoothing (LOESS) regression on all 10-kb windows (dots). Below each plot shown is the Xist locus (green bar) and Xist entry sites (black bars). (F) Correlation between H3K27me3 and H4K20me1 accumulation after 24h of DOX treatment at initially active gene bodies (left), inactive gene bodies (middle) and 10-kb intergenic windows spanning the X chromosome (right). All scales are logarithmic. All correlations (ρ) are with p<0.01 from Pearson's correlation test.
Figure 9Intensity encoded GP-images of the indicated C-Laurdan-stained yeast strains cultured with or without choline, accompanied by the corresponding intensity weighted GP-histogram fitted to a single Gaussian function (light blue line), and transmission image. White arrows point to plasma membranes exhibiting higher lipid packing than internal, organellar membranes; blue horizontal arrows indicate highly packed lipid material in co S(2n-1). To deplete the cho2opi3 parent of PC, cells were transferred to SD C- at OD600 0.1, and cultured into mid-log phase. White scale bars correspond to 10 µm; red scale bars in transmission images correspond to 5 µm. Comparison of average GP-values ±SD from at least 7 different images per strain and condition from two independent experiments, with the datapoints representing the average GP-values of the separate images. Statistical comparison by GraphPad Prism one-way ANOVA: **p<0.01, ****p<0.0001.
Figure 8LAMTOR1 KD significantly reduced levels of GluA1 phosphorylation and total GluA1 as well as the ratio of p-GluA1 to GluA1 in hippocampal neurons, and this effect was reversed by ML-SI1 treatment. A Representative images of CA1 pyramidal neurons stained with anti-p-GluA1 S845 (magenta), anti-GluA1 (red), and anti-GFP (green) antibodies. Scale bar = 20 µm. LAMTOR1 KD significantly reduced levels of GluA1 phosphorylation and total GluA1 as well as the ratio of p-GluA1 to GluA1 in hippocampal neurons, and this effect was reversed by ML-SI1 treatment. B, C Quantitative analysis of the mean fluorescence intensity (MFI) of p-GluA1 S845 (B), GluA1 (C)-immunoreactivity in hippocampal CA1 stratum radiatum (SR) 30 min after LFS. N = 4-6 slices from 4-6 mice. Data information: Data with error bars are represented as means ± SEM. *P < 0.05, **P < 0.01 compared with shSc, #P < 0.05, ##P < 0.01 compared with shLAMTOR1; two-way ANOVA with Tukey's post-test A.U., Arbitrary unit. LAMTOR1 KD significantly reduced levels of GluA1 phosphorylation and total GluA1 as well as the ratio of p-GluA1 to GluA1 in hippocampal neurons, and this effect was reversed by ML-SI1 treatment. , D Quantitative analysis of the ratio of p-GluA1 to GluA1 (D) in hippocampal CA1 stratum radiatum (SR) 30 min after LFS. N = 4-6 slices from 4-6 mice. Data information: Data with error bars are represented as means ± SEM. *P < 0.05, **P < 0.01 compared with shSc, #P < 0.05, ##P < 0.01 compared with shLAMTOR1; two-way ANOVA with Tukey's post-test A.U., Arbitrary unit. E Representative images of proximal dendrites of hippocampal neurons stained with anti-GluA1 (green), anti-LAMP2 (red), and anti-GFP (gray) antibodies. Note that GluA1 labeled with Alexa Fluor 633 secondary antibodies was false-colored green and GFP false-colored gray to better show colocalization of GluA1 with LAMP2. Arrowheads indicate clearly co-localized puncta. Scale bar, 5 µm. F Quantitative analysis of the ratio of the number of GluA1/LAMP2 colocalized puncta to that of total lysosomes in F (n = 17-29 neurons from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. *P < 0.05, **P < 0.01 compared with shSc, #P < 0.05, ##P < 0.01 compared with shLAMTOR1; two-way ANOVA with Tukey's post-test A.U., Arbitrary unit.
Fig.Figure 3 A-L VE-cadherin deletion was initiated either in newborn pups (A, B, E, F) by two applications of 4-OHT at days P2 and P4 or in mice at 11 weeks (C, G) and 42 weeks (D, H) of age (three applications of Tamoxifen at 2 day intervals via oral gavage). Ear skins were prepared 6 weeks (A, B, E, F) or 5 weeks (C, D, G, H) after induction and immunostained for the indicated proteins. Shown are MIPs of confocal tile scans (approx. 750 µm x 750 µm) covering 40 µm in depth. White arrows in (F) highlight distorted and partially fragmented lymphatic vessels and white arrowheads denote aberrantly pointed vessels (F). (I-L) Lymphatic valves in the dermis of the ear were maintained for 5 weeks despite deletion of VE-cadherin at 11 weeks of age (yellow arrows in K, L). PEC1, PECAM1; PRX1, PROX1;VR3, VEGFR-3. Scale bars correspond to 100 μm. The data represent wholemount stainings from 6 (A, E), 5 (B, F), 6 (C, G, K, L), 3 (D, H) and 6 (I, J) analyzed animals
RNA binding and dispersion property of pcMNPs.Figure2RNA binding affinity of pcMNPs analysed by native PAGE; Dispersion stability of pcMNPs in a high-salt solution (50 mM NaCl).
Figure 2Schematic diagram showing Syk-Cn-NFAT pathway axis. Immunofluorescence of Sall4 in reprogramming intermediates treated with DMSO and FK506. n = 5. Scale bar, 100 μm. Concentration (C) and stage (D) test of FK506 during reprogramming. n = 3. RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in reprogramming intermediates treated with DMSO and FK506. n = 3. Diagram showing the procedure of Ppp3ca knockdown at the early stage of reprogramming. RT-qPCR analysis of Ppp3ca expression in MEFs infected with shNC and shPpp3ca viruses. n = 3. Immunofluorescence of Sall4 in WT cells and Ppp3ca knockdown cells on d12. n = 6. Scale bar, 100 μm. RT-qPCR analysis of pluripotent genes expression in cells treated with shNC and shPpp3ca. n = 3. Diagram showing the procedure of shNfatc1 virus infection at the early stage of reprogramming. RT-qPCR analysis of Nfatc1 expression in MEFs infected with shNC and shNfatc1 viruses. n = 3. Immunofluorescence of Sall4 in WT cells and Nfatc1 knockdown cells on d12. n = 3. Scale bar, 100 μm. RT-qPCR analysis of pluripotent genes expression in cells treated with shNC and shNfatc1. n = 3.
Figure 3A Cartoon of constructs containing ChHFR1 or AtHFR1 under the HFR1 promoter of A. thaliana (pAtHFR1) used to complement hfr1-5 mutant of A. thaliana (At hfr1-5). B Relative expression of HFR1 in seedlings of AtWT, At hfr1-5, hfr1>pAt:ChHFR1 (in blue) and hfr1>pAt:AtHFR1 (in red) lines grown under W+FR (R:FR, 0.02). Expression values are the means ± SE of three independent biological replicates relative to the data of 7 days old AtWT. Transcript abundance is normalized to UBQ10 levels. Data information: Different letters denote significant differences (one-way ANOVA with Tukey test, p-value <0.05) among means. C Elongation response of seedlings of the indicated lines grown for 7 days in continuous W or 2 days in W then transferred for 5 days to W+FR (R:FR, 0.02). The mean hypocotyl length in W (HypW) and W+FR (HypW+FR) of at least four biological replicates was used to calculate HypW+FR-HypW. Error bars represent SE. Data information: Different letters denote significant differences (one-way ANOVA with Tukey test, p-value <0.05) among means. D Relative HFR1 protein levels in seedlings of the indicated lines, normalized to actin protein levels, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 line #22 that is taken as 1. Seedlings were grown for 7 days in continuous W (~20 µmol m-2 s-1) after which they were incubated for 3 h in high W (~100 µmol m-2 s-1) and transferred to W+FR (R:FR, 0.06) for 3 h. Data information: Different letters denote significant differences (one-way ANOVA with Tukey test, p-value <0.05) among means.
Figure 4(A) Schematic summarizing glutamine processing in LUAD cells: glutamine uptake by SLC transporters, followed by glutamine-to-glutamate conversion by glutaminase (Gls), followed by glutamate-to-α-ketoglutarate (α-KG) conversion by phosphoserine aminotransferase 1 (Psat1). The chemical inhibitors CB-839 and AOA are indicated. Relative viability of KP and KPΔNF1 clones after CB-839 treatment (IC50: 18 nM) for 72 h assayed via CellTiter-Glo (relative luminescence units). All data points are relative to vehicle-treated controls (n = 4 biological replicates). Relative viability of KP and KPΔNF1 clones after AOA treatment (IC50: 6 µM) for 72 h assayed via CellTiter-Glo (relative luminescence units). All data points are relative to vehicle-treated controls (n = 4 biological replicates). (D) Cumulative population doublings of KP and KPΔNF1 cells incubated with vehicle, 18 nM CB-839, or 6 µM AOA after 6 d in culture (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs). (E) Relative viability of the indicated human KRAS-mutant lung cancer cell lines by trypan blue exclusion assay. Cells were incubated with vehicle, 18 nM CB-839, or 6 µM AOA for 72 h (n = 4 biological replicates). All data points are normalized to vehicle-treated cell lines. For bar charts, data presented as means with error bars representing standard deviations (SDs).
Figure 1A, Quantification of Nox4 mRNA levels (A) in cultured rat cardiomyocytes after increasing degrees of serum starvation as compared to serum-replete cells (15% serum). n=4 cell preparations/group. B Quantification of Nox4 protein levels (B) in cultured rat cardiomyocytes after increasing degrees of serum starvation as compared to serum-replete cells (15% serum). n=4 cell preparations/group. A representative immunoblot for Nox4 and tubulin as a loading control is shown in panel B. C Cell death after 48 h serum starvation in cardiomyocytes in which Nox4 was depleted by an adenoviral shRNA (Ad.shNox4) as compared to cells infected with a control vector (Ad.Ctl). n=6/group. The representative immunoblot shows reduction in Nox4 protein levels after shRNA-mediated knockdown. D Lactate dehydrogenase (LDH) activity in cell supernatants. Staurosporine (Stauro, 1 μmol/L) was used to induce cell death by apoptosis. n=4 cell preparations/group. E Levels of high mobility group box1 protein (HMGB1) in cell supernatants. n=3/group. A representative immunoblot is shown at the top. F Protein levels of cytochrome c in the cytoplasmic fractions of cardiomyocytes. n=4/group. A representative immunoblot is shown at the top. The cellular membrane fraction (Mem) containing mitochondria was used as a positive control. G Percentage of Nox4-depleted and control cells with depolarized mitochondria, quantified by flow cytometry. n=3 cell preparations/group (100,000 cells per experiment). H Effect of cyclosporin A (CsA, 1 μmol/L) on the percentage of cells with depolarized mitochondria. n=6-8/group. I Effect of cyclosporin A (CsA, 1 μmol/L) on cell death in serum-starved cardiomyocytes. n=4/group. J Cell death in serum starved Nox4 knockout MEFs (Nox4KO) and wild-type MEFs (WT). Cat = PEG-catalase (500 U/mL). Nox4KO MEFs were transfected either with active Nox4 or a catalytic inactive Nox4 mutant, Nox4P437H (Mut). n=6-12/group. K HMGB1 levels in the supernatants of serum-starved MEFs in similar experiments to those in (J). n=4/group. A representative immunoblot is shown to the top. L Percentage of MEFs with depolarized mitochondria. n=6/group.
Figure 3(A) A list of the Rad50 mutants used in this study. (B, C) DNA binding analysis for the Interactions between WT or four MjMRcd mutants and a closed circular dsDNA (φx174RFII) in the absence (B) or presence (C) of AMP-PNP/Mg2+. Each protein sample was incubated with φx174RFII dsDNA (3.5 nM) for 30 min on 4°C. The following molar ratio of protein: DNA is used in Fig 3B-3E; lane 2, 50:1; lane 3, 100:1; lane 4, 200:1; lane 5, 500:1; lane 6, 1000:1; lane 7, 2000:1. We note that some MRcd variants may form a second form of the complex with DNA. (D, E) Interactions of the WT or five full-length TmMR mutants with φx174RFII were examined in the absence (D) or presence of AMP-PNP/Mg2+ (E). Reaction conditions are same as those in Fig 3B. (F) Nuclease activities of free MjMre11 (lane 3, 4), nucleotide unbound- (5, 6) and -bound MjMRcd complex (7, 8) towards a hairpin DNA. Each protein sample was incubated with a hairpin DNA (10 nM) in a 1:10 or 1:20 molar ratio (protein: DNA) for 30 min on 55°C. The error bars for the quantified values of uncleaved substrates on the right panel are calculated from the standard deviation from three repetitions of each experiment. (G) Nuclease activities of free TmMre11 alone (lane 3, 4), nucleotide unbound- (lane 5, 6) and -bound (lane 7, 8) full-length TmMR complex towards a hairpin DNA.