EMBO/sd-character-level-ner huggingface dataset:
Fig.Figure 3A In-silico analysis (DECipherment of DNA Elements, SABiosciences) and schematic model of transcription factor binding sites on human FST promoter. CBP: CREB-binding protein; CRE: cAMP response element; TSS: transcription start site. B-C The direct binding of p-CREB to human FST promoter was detected by ChIP-PCR (B) and ChIP-qPCR (C). c-Fos was used as positive control of p-CREB target gene. Normal IgG was used as negative control of ChIP. Data information the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test). D Dual luciferase assay demonstrating the direct binding of pCREB on human FST promoter. pRL-CMV, Renilla luciferase vector; pGL-CRE- and pGL-CRE+, firefly luciferase expression driven by human FST promoter with deleted CRE site (CRE-) or wildtype (CRE+); p-GFPSpark, GFP expressing vector; p-CREBY134F, CREBY134F-GFP expressing vector. Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test). E FST expression and FLT3/ITD signaling were detected by Western blotting in Ba/F3-parental (P in short) and Ba/F3-FLT3/ITD (ITD in short) cells. F-H Phosphor-flow analysis of p-CREB in Ba/F3-parental, Ba/F3-FLT3/ITD, and Ba/F3-FLT3/ITD cells-treated with FLT3 inhibitor Quizartinib (Qui in short). Isotype antibody were used as control to calculate the mean fluorescence intensity (MFI) ratio (F-G). The transcription and expression of Fst was detected by RT-qPCR after Quizartinib treatment (10 nM) in Ba/F3-FLT3/ITD cells for one day (H). Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test). I-K The expression of FST and phosphorylation of CREB were detected by Western Blotting (I and K) and phospho-flow analysis (J) in MOLM-13 (I) and Ba/F3-FLT3/ITD (K) cells treated with Quizartinib and BRD7389 for one day, respectively. Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test). L RSK and FST expression were detected by Western Blotting after p90RSK knockout by CRISPR/Cas9 in MOLM-13 cells. M The phosphorylation of CREB and FST expression were detected by Western Blotting in Ba/F3-FLT3/ITD cells treated with CREB inhibitor 666-15 for one day. ^: non-specific staining of p-ATF1 protein due to the conserved motif. N CREB and FST expression were detected by Western Blotting after CREB knockout by CRISPR/Cas9 in MOLM-13 cells. O The growth of Ba/F3-parental (with IL-3), Ba/F3-FLT3/ITD (without IL-3) and Ba/F3-FLT3/ITD (with IL-3) cells was measured after three days treatment of CREB inhibitor 666-15 in vitro. Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test). P The rescue effect of CREB inhibitor 666-15 on FLT3/ITD-induced dorsalization and axis duplication in zebrafish embryos at 1 dpf.
Co-assembly of M451-90WT with RIPK1497‑583, His-Ub-RIPK3387‑518 and ZBP1170-355 results in the formation of heteromeric fibrils and dense fibrillar networks.Figure 5Representative electron micrographs of homofibrils formed by M451-90, RIPK1497‑583, His-Ub-RIPK3387‑518 or ZBP1170-355 individually, and heterofibrils formed by indicated mixtures.Samples prepared twice from independent protein preparations Heterofibrils containing mCHERRY-M451-90 and YPet‑RIPK1497‑583 C Heterofibrils containing mCHERRY-M451-90 and YPet-RIPK3387‑518 D Heterofibrils containing mCHERRY-M451-90 and YPet‑ZBP1170‑355 E Heterofibrils containing mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑51 Heterofibrils containing mCHERRY- RIPK3387‑518 and YPet‑ZBP1170‑355 Pearson's correlation coefficients measured from two confocal images of the indicated protein mixtures. Heterofibrils containing ECFP-M451-90 with mCHERRY- RIPK1497‑583 and YPet-RIPK3387‑518 STED images of heteromeric fibrils formed by co-assembly of mCHERRY-M451-90 and YPet-RIPK3387‑518
Figure 6(A) Flow cytometry analysis of colonic lamina propria CD11c- CD11b+LY6Chi MHCII-monocytes from steady-state and DSS day 7 treated with TLR2 peptide or scrTLR2 peptide, showing comparable monocytes infiltrates in to the colitic colon. Plots were pre gated on live CD45+ cells. Results are representative of three independent experiments (n=3 per group). (B) Graphical summary of monocytes presented as % out of CD11b + cells.
Figure 3A. Schematic representation of the analysis workflow for the panels (C-F). Optimal and non-optimal transcripts are predicted based on the zebrafish optimality code (Fig 1F) and mRNA decay during the MZT in each species was analyzed. B. Schematic representation of the analysis workflow for the pannels (G-J). The top and bottom quartiles of mRNAs, ranked by stability during the MZT were determined for each species. The codon composition of each group was compared and correlated with the codon optimality defined in zebrafish (CSC). C-F. Cumulative distributions of mRNA fold change for optimal (red), non-optimal (blue), and control transcripts across zebrafish (C), Xenopus (D), mouse (E) and Drosophila (F). G-J. Codon enrichment/depletion between the most unstable (quartile) and the most stable (quartile, n = 1500) maternal mRNAs during the MZT in zebrafish (G), Xenopus (H), mouse (I) and Drosophila (J). Most optimal codons (red: light, weak; dark, strong) are depleted and non-optimal (blue: light, weak; dark, strong) codons are enriched in unstable mRNAs compared to stable ones. P-values are indicated for the comparisons between enrichment/depletion in each species and codon optimality defined in zebrafish (CSC) (Spearman rank test).
Figure 4A WT and R105AD106A mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline, at the same time cells were untreated (UT) or treated with TSQ for 6 hrs. Cells were fractionated into cytosol (C) and crude membrane (M). Fractions were analysed by BN- or SDS-PAGE, Western blot and probed with the indicated antibodies. Representative of three independent experiments. B Cell lysates from (A) were subjected to UBA-pull down and analysed as described above. C WT, Q343A and S345D mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline, at the same time cells were treated ± TSQ for 16 hrs, followed by UBA-pulldown. Representative of three independent experiments. D WT and Q343A mutant MLKL were inducibly expressed in Ripk3-/-Mlkl-/- MDFs by doxycycline, at the same time cells were treated ± TSQ for 16 hrs, followed by UBA-pulldown. Representative of three independent experiments. E HT29 cells were stimulated with TSI, ± NSA (500 nM), or left untreated (UT) for 16 hrs, followed by cellular fractionation. Fractions were analysed by BN- or SDS-PAGE, Western blot and probed with the indicated antibodies. Representative of three independent experiments. F Cell lysates from (E) were subjected to UBA-pulldown and analysed as described above.
Figure 6A) TDP-43 ALS-linked amino acid substitutions K181E, A315T/E, A321G, Q331K, M337V, and A382T. Boxed in the C-terminal domain is the α-helical structure within the conserved region (a.a. 320-343). Droplets formed by TDP-43 wild-type (WT) and the different mutants (4 μM) observed by brightfield microscopy in the presence of no RNA control or A(GU)18 RNA (3.9 μM) at 250 mM NaCl. Mutations A321G and M337V, showing decreased liquidity of the condensates in the presence of A(GU)18, are boxed in red. Representative images for 3 biological replicates using 3 different protein preparations of WT and M337V, 2 preparations of A321G, Q331K, K181E and one preparation of A315T/E and A382T. Scale bars, 10 μm. B) Phase separation of WT (4 μM, 10% Oregon green-labeled protein) and M337V (4 μM, 10% Cy-3-labeled protein) observed by brightfield and fluorescence microscopy in the presence of A(GU)18 (3.9 μM) at 250 mM NaCl. The middle panel shows mixing of these WT (2 μM) and M337V (2 μM) samples. Representative images for 3 biological replicates using 2 protein preparations. Scale bars, 10 μm.
Figure 3A. Current model of ICLpt repair in Xenopus egg extracts (Amunugama et al, 2018). B. pICLpt was isolated via plasmid pulldown at the indicated times after incubation in Xenopus egg extract in the presence or absence of CDC7i (100 µM) and associated proteins were analyzed by immunoblotting. C. pICLpt was replicated in egg extracts in the presence of [α-32P]dATP for the indicated times, and reactions were analyzed by native agarose gel electrophoresis. The p97 inhibitor NMS-873 (p97i; 200 μM), was supplemented to the reaction where indicated. Note that in the absence of p97 activity, CMGs are no longer unloaded from the plasmid, leading to accumulation of replication intermediate products (RI) (Fullbright et al, 2016). OC, open circular; SC, supercoiled. D. Samples from (C) were recovered via plasmid pulldown at the indicated time points as in (B) and analyzed by immunoblotting. E. pICLpt was replicated in mock- or SCAI-C-depleted egg extracts, and samples collected at the indicated time points were analyzed by immunoblotting. F. pICLpt was replicated in mock- or SCAI-depleted egg extract in the presence of [α-32P]dATP for the indicated times, and reactions were analyzed by native agarose gel electrophoresis. ∆SCAI-C and ∆SCAI-N denote SCAI immunodepletion with an antibody raised against the C- or N-terminus of SCAI, respectively; RI, replication intermediates; OC, open circular; SC, supercoiled. G. Schematic of pICLpt illustrating the SapI site, which is regenerated upon replication-coupled repair (Knipscheer et al, 2009). H. Quantification of SapI regeneration in Mock or SCAI-depleted extracts. Note that plasmids containing a SapI site but no ICL account for ~5-7% of each pICLpt preparation. Primary data are shown in Figure EV2F,G. A representative of two independent experiments is shown. I. Schematic of intermediates and extension products generated by AflIII digest of pICLpt. J. pICLpt was replicated in mock- or SCAI-depleted egg extract in the presence of [α-32P]dATP for the indicated times, and reactions were digested with AflIII and analyzed on a denaturing polyacrylamide gel. Stalling points relative to the ICL site are indicated. K. Schematic of intermediates and extension products generated by PsiI+XhoI double digest of pICLpt. L. Samples in (J) were digested with PsiI+XhoI and analyzed on a denaturing polyacrylamide gel. Stalling points relative to the ICL site are indicated. M. Quantification of extensions with deletions in (L) at 240 min (mean±SEM; n=6 independent experiments; **, p<0.01; two-tailed paired t-test).
a–f) Muscle biopsy from subject 4.figf1(a-f) Muscle biopsy from subject 4.1 subjected to electron microscopy (transverse sections). In many fibers, there is material between the layers of basal lamina (a, arrows; scale bar, 500 nm) or overt exocytic vacuoles (b, arrow; scale bar, 2 μm). Some fibers show a single centralized nucleus (c; scale bar, 2 μm). Mitochondria are of variable size and distribution. In some fibers, they form a loop around the nucleus in the periphery of the fiber, resembling a necklace as in c; in others, they form clusters (d; scale bar, 2 μm). The appearance of cristae is often abnormal (e; scale bar, 2 μm; f; scale bar, 500 nm). N, nucleus; M, mitochondrion.
Figure 1A, Hierarchical clustering analysis of RNA-sequencing data from cells treated with DMSO, 20 Gy IR, 250nM AZD6738, or AZD6738 + IR (n=2 biological replicates). B, Top enriched pathway analysis of differentially expressed genes in each cluster C, Heat map of 33 representative inflammatory genes regulated by AZD6738 + IR. D, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, cells were harvested at indicated time points and immunoblotting was performed with indicated antibodies. h, hours; pSTAT1, pSTAT1(Y701); pIRF3, pIRF3(S396); pTBK1, pTBK1(S172). E, MCF10A WT cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with indicated dose. 72 hours later, cells were harvested for immunoblotting with indicated antibodies. F, Same condition as E except the irradiation dose with 20 Gy, mRNA levels of inflammatory genes as indicated were assessed by real-time quantitative PCR, presented as mean + standard error of the mean of three biological replicates.
Apc loss, GSK3 loss and homozygous mutation of β-catenin are sufficient to induce a rapid crypt-progenitor phenotype, but not a single β-catenin mutation.Figure 1Wild-type mice have small defined crypts in the small intestine with little nuclearβ-catenin at the bottom of the crypt. The small intestine of AhCreERAPCfl/fl, AhCreERCatnblox(ex3)/lox(ex3) (day 5) or AhCreERGsk3alphafl/fl betafl/fl (day 6) show the crypt-progenitor cell (CPC) phenotype with increased crypt size (red bar) and nuclearβ-catenin (arrows) along the crypt-villus axis. Scale bar, 100 μm. A heterozygous activation of β-catenin (AhCreER Catnblox(ex3)/+) shows no increase in crypt size or nuclear β-catenin at days 5-10. At day 15 post-induction, accumulation of nuclear β-catenin (arrows) becomes evident with a dramatic CPC phenotype at about day 25. Scale bar, 100 μm. Culture of small intestinal crypts of WT and VilCreER Apcfl/fl (or AhCreER Apcfl/fl) mice with/without R-spo1 shows the dependence of the Wnt agonist in WT organoids but not in Apc-deficient spheres. Representative photos were taken at day 5 in culture. Black scale bar, 50 μm. At day 5 post-induction, only crypts from AhCreERCatnblox(ex3)/lox(ex3) but not AhCreERCatnblox(ex3)/+ survive in culture without the addition of R-spo1. At day 10 post-induction, we observed a mixed phenotype of more organoid-like structures in AhCreERCatnblox(ex3)/+ compared to spheres from Catnblox(ex3)/lox(ex3)crypts in the first week of culture. Black scale bar, 50 μm. Quantification of organoids/spheres at day 10 post-induction (N = 2 or 3 mice per genotype, mean of 6 technical replicates per mouse).
Figure 4A Overview of each patient's precision oncology platform results. The highest tier of therapy options for each patient are shown. A total of 55 recommendations were made in 39 patients B Tier of therapy and related molecular alterations. SNV with LOH refers to SNV in a tumor suppressor gene with LOH. Treatment response by therapy tier. 14 of 29 patients with molecular-based therapeutic options received the treatment. D Treatment response by therapy tier. 14 of 29 patients with molecular-based therapeutic options received the treatment. E Tests contributing to the identification of treatment by tier.
Figure 6(A) Mean percentages of phenotypic HSCs [c-Kit+Sca-1+Lin- (KSL) CD135-CD48-CD150+] in the bone marrow (BM) of 4-month-old controls (Npm1F/FVav1Cre-), Npm1 cKO (Npm1F/FVav1Cre+), p53 KO (Npm1F/Fp53-/-Vav1Cre-) and DKO (Npm1F/Fp53-/-Vav1Cre+), (n = 8 mice per group). (B) Quantification of mitochondrial volume, identified by volume rendering of TOM20, in sorted HSC (n = 83 cells in Ctrl, n = 70 cells in cKO, n = 41 cells in p53 KO and n = 43 cells in DKO, from 3 mice per group). (C) Percentage of cytoskeletal polarized HSCs (n = 47 cells in Ctrl, n = 46 cells in cKO, n = 30 cells in p53 KO and n = 102 cells in DKO, from 4 mice per group). (D) Representative flow plot (left) and quantification (right) of β-galactosidase enzyme activity (β-gal) staining in KSL fraction (n = 4 mice per group). (E) Representative flow histograms (left) and quantification (right) of Annexin V positive cells in KSL fraction (n = 5 mice per group). (F) Levels of Nlrp3 expression (relative quantitation, RQ) in BM-MNCs (4 months old, n = 6-16 mice per group). (G-H) Quantification of IL-1β (G) and IL-23 (H) in serum (n = 4-12 mice per group). (I) NPM1 and P53 status in bone marrow samples of healthy volunteers, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) with multi-lineage dysplasia (MLD) patients. (J) Schematic model of working hypothesis. Npm1 loss causes (1) the premature aging of HSCs and (2) the activation of their mitochondria. This leads to (3) the activation of the inflammasome complex and the consequent (4) aberrant myeloid expansion. The progression of disease (5) is p53-depended. In the Npm1 cKO mouse model (gray), Npm1 loss increases p53 levels that trigger apoptosis and senescence, leading to bone marrow failure. Functional loss of p53 (red, Double KO) escapes these mechanisms, feeding a positive-feedback loop between inflammation and myeloid expansion, which results in blast accumulation and leukemic transformation.
Fig.Figure 7The model presents a non-canonical function for intracellular dopamine in the proliferation of the hematopoietic progenitor population. The progenitor cells during development employ the module of dopamine synthesis (TH and Ddc) and transporter (DAT) to build their levels of intracellular dopamine (iDopamine). In the earlier stages of lymph gland development, specifically at 48 to 72h AEL, this reservoir of iDopamine (as depicted by the red triangle) enables the progenitor pool to transit from S to G2 phase of the cell cycle. Later, as progenitor cells accumulate in G2, the Dop2R mediated dopamine signaling, confers mitotic competency to progenitor cells enabling them to transit from G2 to M phase. Specifically, Dop2R via DAT facilitates the build-up of iDopamine in G2 progenitor cells which poises them to enter mitosis such that in the presence of a mitogenic cue these cells divide. The activation of Dop2R and DAT function is reliant on extracellular dopamine (eDopamine) sensing whose levels in the system are maintained by progenitor based dopamine synthesis (via recycling) and non-autonomously by the PSC cells (which contribute later at 72h AEL). Thus the eDopamine levels support Dop2R signaling and DAT mediated uptake of dopamine by G2 progenitor cells necessary for their sustenance of mitotic competency. This contributes to the overall proliferative capacity of the hematopoietic organ and enables it to achieve its appropriate size. The importance of intracellular dopamine, either via synthesis or its uptake via DAT in governing progenitor cell cycle phasing either at S/G2 or G2/M transition, highlight threshold levels of iDopamine as a novel regulator of cell cycle control.
a, Sections of the thymus and muscle of fed and starved GFP–LC3 mice were stained with anti-GFP Alexa 488 antibody (green) and 4,6-diamidino-2-phenylindole (DAPI; blue).figf1a, Sections of the thymus and muscle of fed and starved GFP-LC3 mice were stained with anti-GFP Alexa 488 antibody (green) and 4,6-diamidino-2-phenylindole (DAPI; blue). Images are representative of n = 6. b, Thymic stromal cells from GFP-LC3 mice were fixed onto glass slides and stained with anti-GFP Alexa 488 and DAPI. Typical autophagy-positive cTECs and mTEChi are shown. The average frequencies ± s.d. of cTECs and mTEChi scoring autophagy positive (that is, ≥5 autophagosomes) in two independent preparations are indicated. Less than 1% of dendritic cells (DCs) and mTEClo were positive. c, Relative distribution of the number of autophagosomes in autophagy-positive cTECs and mTEChi; a.u., arbitrary units.
Model of the quality control of nascent/newly synthesized proteins and thermotoleranceUnder normal conditions (left panel), cells co-translationally fold newly synthesized proteins with high efficiency.Figure 9Under normal conditions (left panel), cells co-translationally fold newly synthesized proteins with high efficiency. A modest fraction of nascent peptides that cannot fold correctly will be ubiquitylated and targeted for degradation. Proteasomal targeting is aided by the HSPA1, which is present in sufficient amounts to prevent the aggregation of damaged nascent peptides that would impede proteasomal clearance. In response to acute heat shock (middle panel), HSPA1 becomes limiting and aggregation of ubiquitylated nascent polypeptides impedes their degradation by the proteasome. Limitation in HSP70 activity leads to inhibition of global protein synthesis through phosphorylation of eIF2α and mechanisms impinging on translation elongation. Simultaneously, activation of HSF1 leads to transcriptional induction and synthesis of HSPA1. In cells rendered thermotolerant through previous heat exposure and recovery (right panel), increased levels of HSPA1, HSPH1, and DNAJB1 provide increased activity of the HHD chaperone complex to facilitate rapid proteasomal clearance. As a result, eIF2α phosphorylation subsides, and cells maintain protein synthesis despite the environmental challenge. Tumors, including esophageal and oral squamous carcinomas appear to gain a survival advantage by upregulating this pathway.
GemC1 cooperates with E2F5 and associates with MCIDAS and FOXJ1 promoter regions.Figure 4A. and B. MCIDAS (A) and FOXJ1 (B) regulatory elements cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas, Geminin and E2F5, as indicated, or an empty vector (-) as a control. Luciferase values were normalized against co-transfected Renilla luciferase and depicted as fold induction over the control empty vector. Data shown are mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction. C. and D. Chromatin immunoprecipitation of MCIDAS and FOXJ1 promoter fragments by GemC1. HEK293T cells were co-transfected with vectors expressing GemC1-GFP (GemC1) or GFP as a control, E2F5 and DP1, together with the regulatory elements of either MCIDAS (C) or FOXJ1 (D). Following chromatin immunoprecipitation, DNA fragments of the MCIDAS and FOXJ1 promoters were detected in immunoprecipitates by qPCR. Data from a representative experiment are shown as fold enrichment of MCIDAS and FOXJ1 promoter fragments in anti-GFP immunoprecipitates over control IgG immunoprecipitates. At least two independent experiments were performed. E-F. Schematic representation of the promoter fragments from the MCIDAS (E) and FOXJ1 (F) regulatory elements assayed by chromatinimmunoprecipitation. For FOXJ1, two putative transcription start sites (TSS) have been proposed and are indicated (see materials and methods).