EMBO/sd-character-level-ner huggingface dataset:
a–d) Left: fluorescence images of SY5Y cells co-expressing HA-p38 (red) and GFP (green) forms of FL (a) or the indicated truncated Sph1 (b–d).f3(a-d) Left: fluorescence images of SY5Y cells co-expressing HA-p38 (red) and GFP (green) forms of FL (a) or the indicated truncated Sph1 (b-d). Cells were treated with lactacystin (12 h) and recovered in the presence or absence of serum. Graphs: percentage of p38 existing alone in Agm or co-aggregating with Sph1 in the forms of Agm or Agg (left), and susceptibility of p38 existing on its own in Agm or co-aggregating with Sph1 proteins in Agm toward serum-stimulated autophagic clearance (right). Values are expressed as folds Agm in the presence of serum and are mean+s.e. (n=3). *Significant versus serum+ condition calculated using Student's t-test. (e) Autophagic susceptibility of Agm formed by p38 and ANK1-p38 fusion proteins. Values are mean+s.e. (n=3). *Significant versus control group calculated using Bonferroni test. See also Supplementary Fig. S6.
Figure 3A WTAP knockout efficiency in 293T cells detected by immunoblotting. B Luciferase activity analyses in wild-type (WT), or WTAP-/- 293T cells, transfected with a luciferase reporter for IFN-β (IFN-β-luc) or ISRE (ISRE-luc) followed by treatments with intracellular poly(I:C), poly(dA:dT), or VSV-eGFP. The results are expressed relative to Renilla luciferase activity. C qRT-PCR analyses to detect the mRNA abundances of IFNB1, RANTES, DDX58, and ISG15 in WT and WTAP-/- 293T cells followed by treatments with intracellular poly(I:C), poly(dA:dT), or VSV-eGFP. D Immunoblot analyses to detect the WTAP knockdown efficiency in A549 cells after transfection with Ctrl or WTAP-specific siRNAs for 48 hrs. E, F qRT-PCR analyses to detect the mRNA abundance of IFNB1 (E), RANTES and ISG15 (F) in A549 cells transfected with Ctrl or WTAP-specific siRNA, followed by treatment with poly(I:C)-HMW at the indicated time points. G, H ELISA analyses to detect IFN-β (G) and CCL5 (H) secretion in supernatants at 16 h. I, J Immunoblot analyses to detect the protein expression of ISGs in shCtrl and shWTAP A549 cells that were stimulated with poly(I:C)-LMW (I) or poly(dA:dT) (J) at different time points with the indicated antibodies. K, L qRT-PCR analyses to detect the mRNA abundances of IFNB1 (K) and RANTES (L) in A549 cells transfected with empty vector (EV) or WTAP-HA plasmid, followed by treatment with poly(I:C)-HMW at the indicated time points. M, N ELISA analyses to detect IFN-β (M) and CCL5 (N) secretion in supernatants
Figure 5(a) Bipartite network of species (left) and metabolites (right) for metabolites that decreased by at least two-fold compared to the abundance of each metabolite at the beginning of the experiment. Colors represent modules containing many overlapping interactions in the network. Network partitioning into modules was performed using BiMat(Flores Metabolites in bold were depleted and secreted by distinct organisms. (b) Bipartite network of species (left) and metabolites (right) for metabolites that increased in abundance by at least two-fold compared to the beginning of the experiment. Metabolites highlighted in bold were depleted or secreted by different species. Colors represent modules containing many overlapping interactions in the network. Network partitioning into modules was performed using BiMat(Flores (c) Scatter plot of the number of consumed metabolites that decreased by at least two-fold compared to the beginning of the experiment vs. the OD600 value of the monospecies culture at the corresponding time point. Error bars represent 1 s.d. from the mean of three biological replicates. (d) Predicted resource utilization interaction network. Each edge represents at least two co-consumed metabolites that decreased by at least two-fold and the edge width is proportional to the number of co-consumed metabolites. Node size is proportional to the total number of consumed metabolites for each species. (e) Predicted metabolite interchange network representing metabolites that were secreted or utilized by distinct organisms based on a two-fold threshold. Arrows point from the source species to the consumer organism. Node size and line width are proportional to the total number of secreted metabolites and number of predicted metabolite interactions, respectively. Species at the top and bottom of the network are primarily producers or consumers, respectively.
Figure 2MCF10A cells were treated with either DMSO, 25uM 4HNE or 5uM RSL3 for 60 minutes. Isolated protein was assessed by immunoblotting for phospho-p38 (Thr180/Tyr182) and total p38 expression. Shown is one replicate of three independent experiments (n=3). MCF10A cells were treated with either EtOH or 25uM 4HNE for 60 minutes. Isolated protein was assessed by immunoprecipitating phospho-p38 (Thr180/Tyr182) and immunoblotting for 4HNE and phospho-p38 (Thr180/Tyr182). Shown is one replicate of three independent experiments (n=3). MCF10A cells were pretreated for 15 minutes with either DMSO or 10uM BIRB, followed by 60 minutes with either DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and BIRB or RSL3 and BIRB. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were pretreated for 15 minutes with either DMSO, EtOH, or 10uM BIRB, followed by 60 minutes with either DMSO, EtOH, 25uM 4HNE, 5uM RSL3, 4HNE and BIRB or RSL3 and BIRB. Isolated protein was assessed by immunoblotting for prominin2, phospho-p38 (Thr180/Tyr182) and total p38. Shown is one replicate of three independent experiments (n=3). MCF10A cells were treated with RSL3 with either DMSO or 10uM BIRB. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.
GOT2 acetylation regulates mitochondrial NADH/NAD+ redox, cellular ATP production, and NADPH homeostasis A The malate–aspartate shuttle, such as LDH, also plays a key role in regulating cytosolic NADH levels in Panc‐1 cells.Figure 4A The malate-aspartate shuttle, such as LDH, also plays a key role in regulating cytosolic NADH levels in Panc‐1 cells. Cells were treated with AOA (1.6 mM, 20 min), oxamate (5 mM, 20 min), and EGCG (0.5 mM, 20 min). The cytosolic NADH level was measured as described in . B, C GOT2 3K acetylation promotes the net transfer of cytosolic NADH into mitochondria. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the NADH level in the cytosol (B) and mitochondrion (C) was determined in cell extracts as described in . D GOT2 3K acetylation regulates mitochondrial NADH/NAD+ redox in the cell. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the ratio of NADH/NAD+ in the mitochondria of cells was measured as described in . E GOT2 3K acetylation promotes ATP production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, ATP production was determined in cell extracts as described in . F GOT2 3K acetylation promotes NADPH production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the ratio of NADPH/NADP+ was determined in cell extracts as described in . G GOT2 3K acetylation promotes GSH production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the GSH/GSSG ratio was determined in cell extracts as described in . H GOT2 3K acetylation suppresses cellular ROS levels. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, ROS was determined as described in in cells under non‐stressed condition or exposed to menadione (50 μM for 30 min). I GOT2 3K acetylation protects cells from oxidative damage. Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins were treated with increasing concentrations of menadione for 3 h as indicated, and cell viability was determined by counting the remaining adherent cells. J, K Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins were seeded in a 6‐well plate, and the cells were maintained under the condition of low glucose (0.5 mM, J) or high glucose (12 mM, K). Culture medium was refreshed every day, and cell numbers were counted every 1-2 days over a period of 7 days. L, M Panc‐1 stable cells in (J, K) were grown under low glucose (0.5 mM) or high glucose (12 mM) for 7 days. The level of histone H3 phosphorylation at Ser10 was determined by Western blot analysis (L). The percentages of living cells and apoptotic cells were determined by flow cytometry as described in (M).
a,b) Production of phagosomal PtdIns(3)P in E.figf7(a,b) Production of phagosomal PtdIns(3) P in E. coli-containing phagosomes of primary peritoneal macrophages transfected with reporter cDNA encoding an eGFP-tagged PX domain of p40phox (p40 PX-eGFP) and treated with DsRed-expressing E. coli (E. coli-DsRed; a) or beads coated with E. coli outer membrane extract (b). Numbers in bottom right corners indicate time (in min). Original magnification, ×60. Right, quantification of fluorescence in microscopy at left. (c) HPLC analysis of the production of PtdIns(3) P in primary macrophages labeled with 3H-tagged myoinositol and treated with beads coated with E. coli outer membrane extract. (d) PtdIns(3) P production in RAW264.7 cells stably expressing SLAM or a mock construct, transfected with eGFP-tagged p40 PX and treated with beads coated with E. coli outer membrane extract. Data are from three combined experiments (a) or are representative of at least three independent experiments (b,d) or two independent experiments (c) with at least 100 beads or bacteria per experiment (error bars, s.d.).
Autophagy protects HSCs from starvation-induced apoptosis.Figure 2Apoptosis levels in cultured WT HSCs (n = 3-5). Results are expressed as percent caspase activation compared to +cytokines conditions. Apoptosis levels in cultured control (Cnt) and Atg12cKO HSCs and GMPs (n = 3). Results are expressed as percent caspase activation compared to +cytokines Cnt HSCs (100%). Absolute number of HSCs in fed and starved Cnt and Atg12cKO mice (n = 4-8 mice per group). Results are expressed as percent of HSCs in fed mice. Data are means ± s.d. Hatching indicates starved conditions. *P ≤ 0.05, **P ≤ 0.01.
a) Quantitative FRAP analysis of the indicated GFP-p38 variants.f7(a) Quantitative FRAP analysis of the indicated GFP-p38 variants. Top: representative images. Middle: time course of changes in relative fluorescence intensity (RFI). Bottom: percentage of proteins in the diffusing fraction and diffusion time (t1/2). Values are mean+s.e. of ten cells analysed over three different experiments. Scale bars, 10 μm (b) Top: schematic showing all lysine (K) residues in ANK1 domain of Sph1 that may serve as potential ubiquitination site(s). Bottom: relative fold difference in the number of Agm formed by WT and the different 'K to R' mutants of ANK1-p38 fusion protein in SY5Y cells maintained in the presence or absence of serum. (c) IBs for HA (top) and GFP (bottom) of total lysates (Input) and GFP immunoprecipitates (IP) from the cell lysates of SY5Y cells co-transfected with the indicated HA-ubiquitin constructs and GFP-p38 or -ANK1-p38 WT or mutant K385R in the presence of lactacystin. (d) Quantification of the levels of each p38 protein ubiquitinated via K63 compared with K48 ubiquitination. Values were corrected by levels of GFP immunoprecipitated. (e) Top: IF for GFP and K63-linked ubiquitin (red) of SY5Y cells expressing GFP-p38 or -ANK1-p38 WT and mutant K385R (green). Arrows indicate colocalization. Bottom: percentage of Agm positive for K63-linked ubiquitin. (f) Quantitative FRAP analysis of GFP-ANK1-p38 K385R and GFP-ANK1-p38 as in (a). Values are mean+s.e. of ten cells analysed over three different experiments. (g) Top: representative images of GFP-ANK1-p38 K385R Agm in SY5Y cells immunostained for Atg14L, Vps34 and DFCP-1. Bottom: percentage of p38 variant and NA Sph1 Agm that colocalized with Atg14L, Vps34 and DFCP-1 under serum− conditions. Values in all graphs represent mean+s.e. (n=3). *Significant versus serum+ condition or p38 Agm. #Significant versus ANK1-p38 Agm. Significance calculated using Bonferroni test. See also Supplementary Fig. S8.
Figure 3Histological evaluation of H&E-stained sections in the labyrinth zone of murine Cdkn2a-/- (n=5 placentas), Cdkn2a-/-;p53-/- (n=5), p53-/- (n=4), and Cdkn1a-/- (n=3) placentas on day E14.5, compared to WT placentas (n=6). (A−E) The labyrinth zone of the WT (A) differs from those of Cdkn2a-/- (B) and Cdkn2a-/-;p53-/- (C) placentas. Black arrows indicate regular-sized nuclei in the WT placenta and polyploidy in the Cdkn2a-/- and Cdkn2a-/-;p53-/- placentas. Blue arrows indicate normal vasculature in the WT placenta and collapsed vasculature in the Cdkn2a-/- and Cdkn2a-/-;p53-/- placentas. Labyrinths of p53-/- (D) and Cdkn1a-/- (E) placentas did not differ from that of the WT placenta. L, labyrinth. Staining for each placenta was performed in triplicates. Histological evaluation of H&E-stained sections in the labyrinth zone of murine Cdkn2a-/- (n=5 placentas), Cdkn2a-/-;p53-/- (n=5), p53-/- (n=4), and Cdkn1a-/- (n=3) placentas on day E14.5, compared to WT placentas (n=6). (F−G) Ki67 immunostaining in the labyrinth zones of murine WT , p53-/- ,Cdkn2a-/-, and Cdkn2a-/-;p53-/- placentas, on day E14.5 (n=3 placentas from each genotype). (F) Representative images of Ki67 staining in the labyrinth zone are shown. Positive Ki67 trophoblast cells are indicated with arrows. L, labyrinth. (G) Proliferation scores (n=3 scores, derived from three placentas of each genotype) based on Ki67 staining show enhanced Ki67 expression in the placental labyrinths of Cdkn2a-/- and Cdkn2a-/-;p53-/- placentas relative to WT. Values are means +SEM of three scores for each genotype. *P < 0.05 by a one-tailed unpaired Student's t-test. Data information: Scale bars, 50 µm. Histological evaluation of H&E-stained sections in the labyrinth zone of murine Cdkn2a-/- (n=5 placentas), Cdkn2a-/-;p53-/- (n=5) placentas on day E14.5, compared to WT placentas (n=6). (H) Representative images of SA-β-gal staining in the labyrinth zones of WT, Cdkn2a-/-, and Cdkn2a-/-;p53-/- placentas on day E14.5. (I) SA-β-gal activity, quantified by ImageJ sofware in Cdkn2a-/- and Cdkn2a-/-;p53-/- labyrinths, is significantly reduced relative to those in WT labyrinth (n>=7 fields of view from each genotype). Values are means + SEM. (***) p < 0.001 by a two-tailed unpaired Student's t-test. Data information: Scale bars, 50 µm.
Model illustrating the role for Dnmt2 in the codon fidelity.Figure 7Model illustrating the role for Dnmt2 in the codon fidelityIn Dnmt2 wild-type cells, C38 tRNA methylation (red circle) promotes the fidelity of tRNA(Glu) and tRNA(Asp) codons, thus resulting in the translation of correctly folded proteins. In Dnmt2−/− cells, the lack of C38 tRNA methylation (gray circle) reduces the capacity of tRNAs to properly discriminate between Asp and Glu codons, leading to decreased translational fidelity, the production of unfolded proteins, and an aberrant phenotype.
Figure 3(A) Cartoon representation of α1-antitrypsin with GSK716 shown in yellow stick format. The five-stranded β-A sheet is in blue. (B) Interactions of GSK716 within the cryptic site with key residues shown in stick format and hydrogen bonds in black dashed lines. (C) Overlay of ribbon representation of protein chains of GSK716 complex (green) with apo protein (purple; PDB id 2QUG (Pearce et al, 2008)) showing the altered position of Trp194 (stick format) that induces a change in conformation of the Gly192-to-Thr203 loop and re-orientation of Tyr244 (stick format). (D) Surface representation of α1-antitrypsin to show propyl and phenyl pocket. (E) Surface representation of α1-antitrypsin to show protein-ligand complement of the substituted phenyl and 2-oxindole rings. (F) Fo-Fc omit density difference map for the ligand contoured at 3 sigma (red denotes negative, blue denotes positive difference). (G) Representative 2Fo-Fc electron density map contoured at 1.0 sigma around the ligand.
Figure 8(A) Depletion of KLF4 significantly enhances the efficacy of Olaparib in killing TNBC cells (4T1). 4T1 cells with stable expression of shKLF4 were implanted into mammary fat pad of BALB/c Nude mice. Drug treatment was started at 10th day. Placebo or Olaparib at the dose of 100 mg/kg was administrated daily for twelve days. Tumor volumes were measured every other day. Tumor volumes were measured every other day. n=9 per group, p values were labeled in figure, one-way ANOVA assay. (B & C) The image of 4T1 xenograft tumors (B) and tumors were weighed and summarized (C). n=9 per group, p values were labeled in figure, one-way ANOVA assay. The heatmap of synergizes score distribution between Olaparib with ABT-263 The synergy score was calculated based on Bliss model according to a model that permits synergistic interaction between Olaparib with ABT-263 The heatmap of synergizes score distribution between Olaparib with dasatinib The synergy score was calculated based on Bliss model according to a model that permits synergistic interaction between Olaparib with dasatinib. The combination effect when combine of Olaparib with ABT-263 and dasatinib in suppression MDA-MB-231 xenograft tumor growth. 8 weeks SCID/Beige female mice were injected 8x106 MDA-MB-231 cells in matrigel (1:1 volume). When tumor reach to 50mm3, 100mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) was administrated by oral gavage daily, 30mg/kg ABT-263 (dissolve in 5%DMSO) was administrated by intraperitoneal injection every other day, 50mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) or placebo (2%DMSO+30%PEG400+saline) were administrated by oral gavage every other day. Mice were treated with single or combination drugs for four weeks. Tumor volumes were measured every other day (F). The image of MDA-MB-231 xenograft tumors The combination effect when combine of Olaparib with ABT-263 and dasatinib in suppression MDA-MB-231 xenograft tumor growth. 8 weeks SCID/Beige female mice were injected 8x106 MDA-MB-231 cells in matrigel (1:1 volume). When tumor reach to 50mm3, 100mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) was administrated by oral gavage daily, 30mg/kg ABT-263 (dissolve in 5%DMSO) was administrated by intraperitoneal injection every other day, 50mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) or placebo (2%DMSO+30%PEG400+saline) were administrated by oral gavage every other day. Mice were treated with single or combination drugs for four weeks. Tumor volumes were measured every other day (G). The tumors were weighed and summarized (H). Placebo (n=7), ABT263 (n=9), Dasatinib (n=8), Olaparib (n=6), Olaparib+ABT263 (n=9), Olaparib+Dasatinib (n=10), p values were labeled in figure, one-way ANOVA assay. (I) Staining of H & E, KLF4, PARP1, p21 and Bax in MDA-MB-231 xenograft tumors. Scale bars, 100 μm. Combination of ABT-263 or dasatinib with Olaparib treatment decrease KLF4 and p21expression, but increase Bax expression in xenograft tumors.
MSR1 ubiquitylation and JNK activation correlates in human tumour associated macrophages (A) Immunohistochemistry analysis of MSR1 in patient ovarian, uterus and sarcoma cancers showing tumour-associated macrophages.Figure 6(A) Immunohistochemistry analysis of MSR1 in patient ovarian, uterus and sarcoma cancers showing tumour-associated macrophages. (B) Pull-downs of K63 polyubiquitin chains and IB analysis of five human primary cancers shows a correlation between the amount of polyubiquitylated MSR1 and JNK activation in an ovarian tumour. (C) Working model: MSR1 is activated by ligation through many different substrates, including apoptotic cells, fucoidan or oxidised LDL (Signal 1). However, only when the macrophage is IL-4-activated (Signal 2) becomes MSR1 ubiquitylated by an unknown E2/E3 ligase. This ubiquitylation recruits Tab2/3, Tak1, Mkk7 and finally JNK, thereby allowing MSR1 to signal directly through the JNK signalling pathway which induces pro-inflammatory gene transcription.
Figure 1(A) The indicated yeast strains (wild type, pex19Δ, pex3Δ, msp1Δ, pex19Δmsp1Δ, and pex3Δmsp1Δ) expressing RFP-SKL (peroxisomal marker) and mitochondria-targeted green ﬂuorescent protein (mGFP) were grown to mid-log phase and imaged by ﬂuorescence microscopy. Representative images are shown, scale bar, 2 μm. DIC, differential interference contrast. Enhanced: the red signal intensity was set to 'best-fit' in ZENPro microscopy software analysis. (B) The indicated yeast strains (wild type, msp1Δ, pex19Δ, pex19Δmsp1Δ, pex3Δ, and pex3Δmsp1Δ) were grown to mid-log phase, back-diluted to OD600=1, and serial dilutions were spotted onto agar plates containing synthetic media supplemented with glucose (S-D), glycerol (S-Gly), glycerol and oleate (S-Gly-Ole), or oleate (S-Ole). (C) Wild type versus pex19Δ RNA levels. Outliers are indicated. Peroxin genes are indicated in red. The blue shading emphasizes that the RNA levels of the indicated genes are lower in pex19Δ compared to wild type. (D) Cumulative distributions of fold changes (log2) in RNA between wild type and pex19Δ yeast (Kolmogorov-Smirnov test). Peroxin genes are indicated in red. (E) Translational efficiency (TE) of wild type versus pex19Δ. Outliers are indicated. Peroxin genes are indicated in red. The red shading emphasizes that the translation efficiency of the indicated genes is higher in pex19Δ compared to wild type. (F) pex19Δ versus pex19Δmsp1Δ RNA levels. Outliers are indicated. Peroxin genes are indicated in red. The red shading emphasizes that the RNA levels of the indicated genes are higher in pex19Δmsp1Δ compared to pex19Δ. (G) Cumulative distributions of fold changes (log2) in RNA between pex19Δ and pex19Δmsp1Δ yeast (Kolmogorov-Smirnov test). Peroxin genes are indicated in red. (H) Translational efficiency (TE) of pex19Δ versus pex19Δmsp1Δ. Outliers are indicated. Peroxin genes are indicated in red. The blue shading emphasizes that the translation efficiency of the indicated genes is lower in pex19Δmsp1Δ compared to pex19Δ.
Effects of lentivirus-mediated shRNA knockdown of the FGL2 and CFTR genes in intestinal organoids.Figure 6A) Schematic of patient-derived intestinal organoid acquisition and expansion followed by two functional assays. B) qPCR Validation of lentivirus-mediated shRNA knockdown of FGL2 and CFTR in intestinal organoids. Significance of sample differences was calculated using the one-tailed student t-test (n=2 biological replicates). All values are mean ± s.d. EV = empty vector. C) Representative confocal microscopy images of calcein green labelled FIS organoids of a healthy individual and a CF patient, before and 60 min after stimulation with Fsk (5 uM). Scale bar, 200 μm. D) Quantification of FIS response in wt-CFTR organoids (ORG-01(HC)) shown in (C) as area under the curve at 60 min. Significance of sample differences was calculated using two-way ANOVA (n=2 biological replicates). All values are mean ± s.d. E) Quantification of FIS response in F508del-CFTR organoids (ORG-BX-002 (dF/dF)) shown in (C) as area under the curve at 60 min. Significance of sample differences was calculated using two-way ANOVA (n=2 biological replicates). All values are mean ± s.d. F F) Tracing of Ussing chamber measurements of the transepithelial current produced by 2D monolayer cultures of specified organoids. The F508del-homozygous monolayers with the knockdowns have been rescued with Trikafta.