EMBO/sd-character-level-ner huggingface dataset:
ETC-159 synergizes with the PARP inhibitor Olaparib A, B.Figure 1A, B. PORCN inhibitor ETC-159 synergizes with PARP inhibitor olaparib in suppressing proliferation of HPAF-II pancreatic cancer cells in soft agar. The ED50 (dose that reduced colony formation by 50% of the maximal inhibition) was determined (Table EV1) and cells were treated with ETC-159, olaparib or the combination at the indicated dose (for example 0.25 x ED50 of Olaparib or ETC-159, or 0.25 x ED50 of Olaparib + 0.25 x ED50 of ETC-159, respectively). (A) The data is representative of two independent experiments with each point representing an average colony count ± SD of duplicates. (B) The Combination Index (CI) values of olaparib and ETC-159 calculated for the two independent experiments using the Chou-Talalay CompuSyn software. CI < 1, = 1, and > 1 indicate synergism, additive effect, and antagonism, respectively. The lower the CI, the stronger the synergism. C, Olaparib and ETC-159 synergize to prevent the growth of HPAF-II xenografts in mice. NSG mice with established HPAF-II subcutaneous xenografts were randomized into four groups. Mice were gavaged daily with ETC-159 (10 mg/kg), Olaparib (50 mg/kg) or a combination of ETC-159 (10 mg/kg) and Olaparib (50 mg/kg). Treatment was initiated after HPAF-II tumors were established. (C) Tumor volumes were measured starting from day 0 and during the course of treatment as shown. Data points represent the mean ± SD. n = 7-8 tumors/group. p-values indicate significant difference compared to the vehicle group. D. Olaparib and ETC-159 synergize to prevent the growth of HPAF-II xenografts in mice. NSG mice with established HPAF-II subcutaneous xenografts were randomized into four groups. Mice were gavaged daily with ETC-159 (10 mg/kg), Olaparib (50 mg/kg) or a combination of ETC-159 (10 mg/kg) and Olaparib (50 mg/kg). Treatment was initiated after HPAF-II tumors were established. (D) Tumor weights in the respective groups at the end of 21 days treatment are shown, each dot represents an individual tumor and line represents mean. p-values were calculated with Mann-Whitney U test. E, F. Olaparib and ETC-159 synergize in multiple Wnt-addicted cancer cells. Soft agar colony formation assays were performed as in Figure 1A with the indicated cell lines treated with varying concentrations of ETC-159, olaparib or a combination of both as indicated and the combination index calculated from two independent experiments using Chou Talalay method are shown. PARP inhibitor olaparib and ETC-159 synergistically prevent colony formation of EGI-1, MCAS and CFPAC-1 cells in soft agar. (F) Representative image of soft agar colonies of EGI-1 cells is shown.
Cys 81 is required for redox regulation of HsAtg4A in cells.Figure 8(A) CHO cells stably expressing GFP‐GATE‐16 were transiently transfected with HsAtg4AWT‐Myc‐His6 or HsAtg4AC81S‐Myc‐His6. At 24 h post‐transfection, cells were starved for 2.5 h after which they were fixed, permeabilized and incubated with anti‐Myc monoclonal antibodies. Upper panel: typical images of cells transfected with the above constructs, as visualized using a confocal microscope; representative autophagosomes are indicated by arrows. Lower panel: quantification of the average number of autophagosomes per cell in the different transfectants. Images of the fixed cells were visualized using a Nikon eclipse TE300 fluorescent microscope and used for quantification of the number of autophagosomes per cell. The results presented are the means±s.d. of a total of 100 cells from three separate experiments. (*) indicates significance at P0.001. (B) Left panel: HEK 293 cells were cotransfected with GFP‐GATE‐16 and each of the Atg4A constructs mentioned in (A). At 40 h post‐transfection, the cells were starved for 2.5 h, lysed in Ripa buffer and 100 μg of each lysate was loaded to 10% SDS-PAGE and subsequently analyzed with anti‐GFP antibodies to detect the transfected GATE‐16, anti‐Myc antibodies to detect the transfected HsAtg4A and anti‐tubulin antibodies as control. (*) indicates non‐lipidated GFP‐GATE‐16 and (**) indicates lipidated GFP‐GATE‐16. Right panel: results from three separate experiments, as detailed in the left panel, were analyzed using NIH image program and quantified as follows: the amount of lipidated GFP‐GATE‐16 out of the total of GFP‐GATE‐16 was calculated for each mutant in each experiment. The value obtained for HsAtg4AWT in each experiment was set to 100% and the relative lipidation in cells transfected with the mutant was calculated accordingly.
Figure 4A Extracellular flux analysis by measuring oxygen consumption rate (OCR) (left panel) and extracellular acidification rate (ECAR) (right panel) in LNCaP cells indicates that SDHA/SDHB silencing increased both activities compared to ENZA alone. B. Comparison of OCR vs ECAR clearly shows significant upregulation of glycolytic capacity (2 fold) in SDH-repressed cells compared to ENZA alone.. C. Silencing of SDHA/SDHB subunits in LNCaP cells increased p-AMPK, as well as p-p38/p-Hsp27 levels. Co-silencing of AMPK inhibited the upregulation of AR mediated by SDH repression. D. SDHA and SDHB overexpression decreased p-CaMKK2 and p-AMPK levels in LNCaP cells. E. SDHA and SDHB silencing increased AR, p-CaMKK2 and p-AMPK axis in LNCaP cells, which was inhibited by CaMKK2 silencing. F.. Treatment with 5 µM STO-609 for 24 h in LNCaP cells inhibited AR upregulation induced by SDHA/SDHB subunit silencing. G. Thapsigargin, DMM, DMS and ENZA treatment for the indicated time points increased intracellular calcium flux in LNCaP cells as visualized with calcium sensitive Fluo4-AM probe under confocal microscope.
Figure 1Paneth cell quantification on Lgr5-DTReGFP duodenum (Duo) and Ileum (Ile). Left panel: Representative images of Lendrum's staining that evidence Paneth cell granules. Arrows show differentiated Paneth cells. Right panel: quantification of the Paneth cells per 10 intervilli regions. Each symbol indicates the value for a given embryo. Expression analysis by qRT-PCR of the indicated Paneth cell markers in Lgr5-DTReGFP ileums (n= 8 WT, 12 HE, 10 KO). X-gal staining in Axin2Lac/+-Lgr5-GFP-CreERT2 WT or KO ileums. Gene expression analysis by qRT-PCR of stem cell markers in Lgr5-DTReGFP ileums (n= 3 WT, 6 HE, 6 KO). FACS quantification of the number of eGFP+ve (ISC) cells per small intestine at the indicated developmental stages in Lgr5 HEs and Lgr5 KOs. Each symbol indicates the value for a given embryo. Mean value for each group is depicted on the graph. Plating efficiency of ex vivo cultured E18.5 small intestines quantified 6 days after initial seeding. Each symbol indicates the value for a given embryo. Influence of mouse Rspondin 1 concentration on growth of replated Lgr5-DTReGFP WT and Lgr5 KO organoids after 5 days of culture.
Viral propagation and quantitation.Figure 2.A) Two SARS-CoV-2 passage 4 stocks (black and gray) were quantified utilizing plaque assay at day two (closed circles) and day three (open circles) post infection of Vero E6 and Vero CCL81. B) Plaque morphology on Vero E6 and Vero CCL81 at day 2 and day 3 post inoculation. C) Cell monolayers two days post infection in Vero E6 (top) and Vero CCL81 (bottom row) at multiple dilutions.
a, Thymi from wild-type (WT) or Atg5–/– embryos were transplanted under the kidney capsule of wild-type recipients and analysed 6 weeks later.figf2a, Thymi from wild-type (WT) or Atg5-/- embryos were transplanted under the kidney capsule of wild-type recipients and analysed 6 weeks later. The average cellularities ± s.d. are shown; n = 5. b, Normal compartmentalization of Atg5-/- grafts in cytokeratin-8-positive (red; K8) cortical and cytokeratin-5-positive (green; K5) medullary regions; n = 8. c, Expression of MHC class I and II on cTECs or mTECs from Atg5-/- to wild-type (WT) grafts (red), or wild-type to wild-type grafts (blue). Staining controls are depicted in grey; data are representative of three experiments. d d, RT-PCR analysis of autoimmune regulator (Aire) and TRA expression in mTECs isolated from Atg5-/- to wild-type, or wild-type to wild-type grafts. Expression of β-actin (Actb), intestinal trefoil factor (Tff3), casein 2 (Csn2) and salivary protein 1 (Spt1) are also shown (5-fold serial dilutions). Data are representative of two experiments.
Figure 3A-C. Average tumor volumes of xenografts derived from H1915 (A), H650 (B) and (C) H1915 cell line with doxycycline inducible ABL1 gate-keeper mutation (H1915-ABL1T315I) in mice treated with either vehicle or imatinib (100mg/kg). Error bars represent ±S.D., n=6-10 mice per group, *P=0.0016 by Mann-Whitney U test.
Figure 3(A) Structure of the TRIM32 RING dimer in ribbon representation with each RING monomer coloured in cyan and blue and the Zn2+ ions as grey spheres. (B) Overlap of the RING dimers of TRIM32 (cyan, 5FEY.pdb), TRIM25 (yellow, 5FER.pdb) and TRIM5 (orange, 4TKP.pdb). The structures were overlapped on the circled RING domain. This overlap shows that the structures of the RINGs are very similar but that there are differences in the relative orientations of the two RINGs. (C) Structure of the TRIM25 RING/E2~Ub complex with the RING domains in the same colour scheme as in (A), UBE2D1 in grey and the ubiquitin molecules in salmon and red.
Figure 9Principal component analysis of exRNA, transcriptome, proteome, and clinical covariate data from samples collected at the first timepoint. The first two components were used to describe the distribution of samples based on expressed genes, proteins and clinical data, respectively, whereas the first and third components were used for samples based on the exRNA data. Performance of prognostic models based on exRNA, transcriptome, proteome, and the corresponding clinical covariate data sets. Model performance of the five-fold cross-validation was assessed using the Matthews correlation coefficient (MCC), AUC, accuracy, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). Data were represented as means ± SD. The most frequently selected features of exRNA-, transcriptome-, proteome-, and clinical-based models. Features were simultaneously identified from each of the four data sets and for each of the four machine learning algorithms based on the frequency of variables used by AI models during 50 runs of the five-fold cross-validation. Correlation heatmap among the most frequently selected features (frequency > 0.78) used in the exRNA-based model. Members in the let-7 family selected for the exRNA-based predictors (hsa-miR-98-5p, hsa-let-7a-5p, hsa-let-7d-5p, hsa-let-7f-5p) were highly correlated with each other. (E-I) Biomarkers identified from exRNA (E), transcriptome (F), proteome (H) and clinical data (I) based models exhibited a clear separation between those patients with either good or poor prognosis. (G) Functional enrichment of 110 protein features selected from random forest modeling (Fisher-extract test, p < 0.05).
Figure 3Bioluminescence images visualize mock and carboplatin treated OVCAR4 xenograft tumors (day 53; after 13 d i.p. treatment). Color scale unit: p/sec/cm2/sr. Charts illustrate the tumor growth (B) and ascites volumes at the end of the follow-up (C). Carboplatin treatment started on day 35. Bioluminescent unit: p/sec/cm2/sr. N = 5 mice/group. Representative micrographs of hematoxylin-eosin staining in the xenografts tumors. Scale bars: 100 μm EphA2-pS897 immunofluorescence in the xenografts tumors. Scale bars: 10 μm Quantitative assessment of tumor EphA2 (F) and EphA2-pS897 (G). N = 5 mice/group. Confocal micrographs of tumor EphA2-pS897 (red) and cleaved caspase-3 (H; clCasp3, green) and corresponding clCasp3 quantification (I). Scale bars: 20 μm. N = 5 mice/group.
a) Differential interference contrast micrographs of root epidermal cells incubated for 8 h with concanamycin A, in nutrient starvation conditions.f2(a) Differential interference contrast micrographs of root epidermal cells incubated for 8 h with concanamycin A, in nutrient starvation conditions. Autophagic bodies accumulation in the vacuole is clearly visible in wild-type seedlings, absent in the atg5-1 mutant and restored in atg5-1 seedlings expressing ATG5-GFP. (b) Confocal microscopy. The ATG5-GFP fluorescence pattern in the atg5-1 background is identical to that observed in wild-type seedling, including ring-like structures. Scale bar, 15 μm.
The antiviral activity of LY6E is evolutionarily conserved and specific to CoVs.Extended Data Figure 1.
Figure 7(A) Immunoblot of Flag-p65-linked ubiquitination promoted by Myc-CDC20. HEK293T cells were co-transfected with Flag-p65, HA-Ubiquitin, with or without Myc-CDC20 or Vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection. (B) Immunoblot of Flag-p65-linked ubiquitination promoted by HA-APC11. HEK293T cells were transfected with Flag-p65, His-Ubiquitin, with or without HA-APC11 or Vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection. Immunoblot of Flag-p65-linked in vitro ubiquitination promoted by GST-CDC20 (C) were incubated with purified proteins, including HA-ubiquitin, E1, E2, and Flag-p65 as indicated at 32 ℃ for 1 h. D) Immunoblot of Flag-p65-linked in vitro ubiquitination promoted by GST-APC11 (D). Bacterially expressed and purified GST-APC11 were incubated with purified proteins, including HA-ubiquitin, E1, E2, and Flag-p65 as indicated at 32 ℃ for 1 h. (E) Immunoblot of Flag-p65-linked ubiquitination promoted by truncated Myc-CDC20 fragments. HEK293T cells were transfected with Flag-p65, HA-Ubiquitin, with or without Myc-CDC20 171-499 fragment (containing WD40 domain), Myc-CDC20 1-170 fragment (WD40 domain deficient) and Vector plasmids. Transfected cells were then treated with 10 μM MG132 for 6 h before collection. (F) Immunoblot of Flag-p65-linked ubiquitination promoted by HA-APC11 in NC and CDC20sh cells. NC or CDC20sh HEK293T cells were co-transfected with Flag-p65, His-Ubiquitin, with or without HA-APC11 and Vector plasmids. Transfected cells were treated with 10 μM MG132 for 6 h before collection.
SOX2 and OCT4-dependent changes in chromatin accessibility A Example tracks of the ATAC-seq signal in SHOH, SHOL, SLOH and SLOL populations for loci where chromatin accessibility is unaffected, affected by OCT4 levels, SOX2 levels, or affected by both.Figure 4Example tracks of the ATAC-seq signal in SHOH, SHOL, SLOH and SLOL populations for loci where chromatin accessibility is unaffected, affected by OCT4 levels, SOX2 levels, or affected by both. Heatmap of relative chromatin accessibility in SHOH/SHOL/SLOH/SLOL samples for all loci with significantly altered accessibility, with examples of genes close to affected regulatory regions. Groups are defined by their significance (OHvsOL/SHvsSL/SHOHvsSLOL) and fold-change direction. Gene Ontology (GO) term enrichment analysis for genes close to regions with higher accessibility in OCT4-high cells (Upregulated OCT4). Chromatin accessibility ratio of SHOH vs SLOL cells in loci overlapping ES cell super-enhancer regions or not. Percentage of regions in each group showing an overlap with OCT4, SOX2 and H3K4me3 ChIP-seq peaks.
Figure 2(A) The interaction between EPCR1 and ARID2 or ARID3. Arabidopsis plants carrying EPCR1-Flag and ARID2-Myc or ARID3-Myc transgenes were used for co-IP. (B) The interaction between TRB1 and ARID2. Arabidopsis plants carrying TRB1-Flag and/or ARID2-Myc transgenes were used for co-IP. (C) The interaction between TRB1 and EPCR1. Arabidopsis plants carrying TRB1-Flag and/or EPCR1-Myc transgenes were used for co-IP. (D) The diagram of protein-protein interactions that were detected in yeast two-hybrid assays. A line represents an interaction between two proteins. If a protein was indicated to interact with another protein when it was fused with both GAL4-BD and GAL4-AD, both straight and curved lines are shown between the two proteins. A lariat loop indicates that a protein can form a homodimer. (E) The interaction between TRB1 and PWWP2 or ARID2 was detected by an GST pull-down assay. (F) The interaction between EPCR1, PWWP2, ARID2, and TRB1 was detected by an HIS pull-down assay. The full-length PWWP2, ARID2, and TRB1, and the EPCR1-N terminal (1-500 aa) were used for the interaction assay. (G) The diagram indicates the interactions among EPCR1, PWWP2, ARID2, and TRB1 as determined by pull-down assay, yeast two-hybrid, and co-IP. The solid line and the broken line indicate, respectively, direct and indirect protein-protein interactions.
Figure 7Figure 7. Structure of a helix-7 Ago2 mutant. A) Superposition of wild type (white) and MI-AA (gray) Ago2. Helix-7 colored yellow; ordered region of the guide RNAs colored in red. B) Close-up view shows helix-7 tilts away from the seed region in the MI-AA mutant. C) Side-by-side comparison of the seed region in wild type (left) and MI-AA (right) Ago2 crystal structures.
Bi-allelic double knockout of miR-23~27~24 clusters in ESCs by CRISPR/Cas9.Figure 6Genomic location and sequence alignment of mouse miR-23~27~24 cluster miRNAs. Black box indicates the seed region. Summary of CRISPR/Cas9-mediated gene targeting in ESCs. Genomic PCR of the specific fragments spanning miR-23~27~24 clusters. WT indicate wild-type ESCs; DKO indicate miR-23a~27a~24-2-/-, miR-23b~27b~24-1-/- ESCs; KO indicate miR-23a~27a~24-2-/- ESCs. Real-time PCR analysis of mature miR-23a/b, miR-27a/b and miR-24 expression in wild-type, DKO and KO ESCs. Data are shown as mean + s.d. (n=3). AP staining of wild-type and miR-23~27~24 cluster knockout ESCs. Real-time PCR analysis of Oct4, Sox2 and Nanog expression in wild-type and miR-23~27~24 cluster knockout ESCs. Data are shown as mean + s.d. (n=3). Oct4, Sox2 and Nanog immunofluorescence staining of wild-type and the knockout ESCs.
Figure 8Ubiquitination of the NB domains from SUMM2 (A), RPP4 (B) and CHS1 (C). Immunoblot of bacterial lysates form E. coil strains expressing AtUBA1-S, MBP-NB-HA, AtUBC8-S, HIS-FLAG-UBQ10 and SNIPER1-myc, or strains without one of these components. Two biological repeats were carried out with similar results.
Combining mycolactone with BZ delays MM tumor growth in vivo.Figure 7A C57BL/6 mice (N=2/group) were injected intraperitoneally with BZ (0.5 mg.Kg-1), BZ (0.5 mg.Kg-1) + mycolactone (0.3 mg.Kg-1), DMSO as vehicle controls every 3.5 days, or left untreated. Blood samples were analyzed 5, 15, 20 and 25 days after the first injection and percentages of CD3+ and CD19+ cells determined by flow cytometry. Results are expressed as % of positive cells among total mononuclear cells, each dot representing the Mean % from 2 mice. B NSG mice (N=9/group) were injected subcutaneously with 3.106 MM.1S cells at day 0. Seven days later, mice were injected with DMSO, BZ (0.5 mg.Kg-1) and/or mycolactone (0.3 mg.Kg-1) every 3.5 days intraperitoneally and tumor growth was followed by daily measurement of the tumor diameter. Data are Mean tumor diameters ± SD and represent cumulative data from 3 independent experiments with 3 mice/group. Difference between groups were analyzed with Tukey's multiple comparison test using Two-way Anova, with mixed effect model for each time points: *p<0.05; **p<0.01; ***p<0.001 (exact p-values indicated). Only significant differences are shown.
Model of sgRNA integration in endogenous ABCB1 locusWhen a lenti-guide Puro vector is used to deliver a gRNA to induce a DSB, the gRNA can be integrated into the break site.Figure 4When a lenti-guide Puro vector is used to deliver a gRNA to induce a DSB, the gRNA can be integrated into the break site. The DSB was induced in the promoter of ABCB1 and therefore the highly active promoters of the vector were driving the expression of the ABCB1 gene. In this case, we were selecting for cells that upregulated ABCB1 and therefore the frequency of this event was higher.
hTauAT expression enhances extracellular glutamate, cytotoxicity and progressive neuronal loss in the hippocampus.Figure 5A. Glutamate content in the culture medium ([Glu]e) from either hTauAT (n=10 culture inserts containing 6 slices each) or control slices (n=5-12 culture inserts) analyzed at DIV 5, 10, 20 and 25. Glutamate content from medium cultured with hTauAT slices is shown in percent of control at each time point. Increased levels were observed between DIV 5 and DIV 20. At DIV 25 glutamate levels already declined to control levels. One-way ANOVA followed by Tukey's post-hoc test *p<0.05; **p<0.01 ***p<0.001.B. Lactate dehydrogenase (LDH) release (a measure of cytotoxicity) analyzed in hTauAT (n=17-19 culture inserts containing 6 slices each) or control slices (n=15-16 culture inserts) at DIV 5, 10, 20 and 25. LDH release of hTauAT slices is shown in percent of control at each time point. A pronounced increase (~25%) in LDH release was observed due to hTauAT expression starting at DIV 10 but not at DIV 5 One-way ANOVA: *p<0.05; **p<0.01 and ***p<0.001. C. Slice cultures from control (left), hTauAT (middle) or hTauAT treated with ceftriaxone (CEF; 100 µM; right) stained against neuronal nuclear protein (NeuN) at DIV 30. Representative neuronal cell layers are shown. hTauAT slices show neuronal loss in the granular cell layer of the dentate gyrus and the pyramidal cell layer in the region of the hilus in area CA3 (middle panel, red arrows). Treatment with ceftriaxone for 30 days prevents neuronal loss in DG and area CA3.D. NeuN positive cell bodies were counted in the DG and within the pyramidal cell layer in area CA3 and CA1 at DIV 30 in defined regions of interest (ROI). The number of neurons was reduced in hTauAT slices, both within area DG and CA3, whereas the number of neurons in the CA1 was only slightly affected. Neuronal loss was largely prevented in cultures treated with ceftriaxone (CEF) in dentate gyrus and area CA3 (n=14-19 slices per group and area; prepared from at least 6 animals). One-way ANOVA followed by Tukey's post-hoc test *p<0.05; **p<0.01
A cross-tissue survey of ACE2+TMPRSS2+ cells in published single-cell datasets.Extended Data Figure 1.
a) Binding of Flag-tagged wild-type LC3B and LC3BI35A, LC3BF52A or LC3BG120A with endogenous C18-ceramide (C18-Cer), generated in response to wild-type CerS1 induction (left) or exogenous C18-Pyr-Cer (right), was measured using LC/MS/MS after pulldown using anti-Flag–conjugated beads.figf5(a) Binding of Flag-tagged wild-type LC3B and LC3BI35A, LC3BF52A or LC3BG120A with endogenous C18-ceramide (C18-Cer), generated in response to wild-type CerS1 induction (left) or exogenous C18-Pyr-Cer (right), was measured using LC/MS/MS after pulldown using anti-Flag-conjugated beads. Data shown are an average of at least three experiments ± s.d. (*P 0.05). (b) Effects of CerS1 and C18-ceramide induction on the lipidation of Flag-tagged wild-type, LC3BI35A, LC3BF52A and LC3BG120A proteins were examined by western blotting. Noninduced cells (− tet) were used as controls. β-actin was used as a loading control. Full blots can be found in Supplementary Figure 13.