EMBO/sd-character-level-ner huggingface dataset:
TGFβ promotes proline synthesis from glutamine for collagen production.Figure 2Comparison of the amino acid representation in all human proteins and all collagens. The x-axis denotes individual amino acids shown by single letter code. Glycine and proline representation in protein acid hydrolysates of untreated NIH-3T3 cells and their secreted ECM, produced as in Figure 1d. Amino acids were measured by GC-MS. Tracing of [U-13C] L-glutamine ([U-13C] Gln) into indicated metabolites. NIH-3T3 cells were treated with TGFβ (2 ng/mL for all experiments) or mock for 48h, and the medium was replaced (including all treatments) with DMEM lacking L-glutamine and supplemented with [U-13C] Gln for the last 8h. Metabolic steady state was reached at this time point (see kinetic labeling curves in Figure EV3b). Metabolites were measured by LC-MS. The left graph shows the total pool size normalized to the packed cell volume; the right side shows the percent labeling. αKG, alpha-ketoglutarate; AU, arbitrary units. Glutamine flux into glutamate, αKG and proline in NIH-3T3 cells treated with TGFβ or mock for 48h, relative to mock-treated cells. Abundance of indicated metabolites in NIH-3T3 cells treated with TGFβ or mock for 48h, relative to mock-treated cells. Metabolites were measured by GC-MS. Schematic of [U-13C] Gln tracing into proline in collagen I-α1 (CO1A1) in NIH-3T3 cell-derived ECM. Expected m/z of unlabeled (m+0) and fully labeled proline (m+5 for one proline, m+10 for two prolines) for the analytical peptide are shown. Representative MS2 spectra of the CO1A1 peptide in ECM generated with unlabeled ([12C] Gln, left) and fully labeled glutamine ([U-13C] Gln, right). Peaks representing fully labeled (m+5, m+10) proline are highlighted in red. Peptides were measured by LC-MS. Relative abundance of the analytical peptide containing unlabeled (m+0) or one/two fully labelled prolines (m+5/m+10) from the CO1A1 peptide in ECM derived from NIH-3T3 cells treated with TGFβ or mock in the presence of fully labelled L-glutamine ([U-13C] Gln). Peptides were measured by LC-MS. The amount of ECM analyzed was normalized to the packed cell volume of cells grown on a parallel plate under identical conditions.
Figure 8(A) Native MS spectrum of 30 µM SPOP28-359 confirms that SPOP self-associates through addition of dimers. The increase in mass indicates that the dimer is the stable building block within the assemblies. (B) Graphical representation of the SPOP concentration of each oligomeric species within a 27 µM SPOP28-359 sample. The cartoons' sizes are scaled based upon the fraction present within the total sample. (C) Concentration-dependence of the SPOP oligomer size distribution (mole fraction) from isodesmic modeling of CG-MALS data.
| VxrAB controls genes involved in iron homeostasis.Figure 4(A) Venn diagram showing overlap between the VxrB regulon and known V. cholerae Fur-controlled genes. (as per RegPrecise3.0 DB, http://regprecise.lbl.gov/RegPrecise/). 20 out of 47 Fur-regulated genes also have a VxrB binding site and are differentially regulated by VxrBD78E. (B) Genomic organization of iron uptake systems and their putative Fur/VxrB binding sites. (C) Time-course of iron accumulation in PenG-treated WT and ∆vxrAB cells as measured by ICP-MS. Data are average of 3 independent biological replicates, error bars represent standard deviation. ∗∗P=0.001; ∗∗∗P<0.0003 (paired t-test). (D) Streptonigrin-sensitivity of PenG-treated cells. Cultures were grown to a density of ~2 x 108 cfu/ml. Before ("- PenG") and after addition of PenG (100 µg/ml, 10x MIC), ~ 5 x 107 cells were spread on an LB agar plate. In the PenG-treated samples ("+ PenG"), beta lactamase solution was also added to remove the antibiotic. Streptonigrin (5 µL of 5 mg/ml, in DMSO) was then added on a filter disk, followed by incubation for 24 h at 37 ˚C. Values represent average diameter of clearance zones (+/- standard deviation) of six biological replicates. SNG, streptonigrin.
Figure 3Distribution of gene-associated and orphan reactions in the six modules defined in iJL208. Distribution of genes and their associated final annotation score (see Figure 1F) in the metabolic modules. JCVI-syn3.0 orthologs are plain while the non-orthologs are hatched.
MVP impairs the recruitment of autophagy markers.Figure 5A. Impaired recruitment of p62 to MVP positive Listeria. HeLa cells were transfected with MVP-GFP (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy, and stained with phalloidin (blue) and anti-p62 antibody (red). Inset regions are magnified. Arrows indicate independent bacteria The scale bar represents 1 µm. The vast majority of MVP-positive bacteria were completely devoid of anti-p62 labeling (95.1±2.0%; mean ± SEM from n = 3 experiments) but 4.9±2.0% (mean ± SEM from n = 3 experiments) were stained at one pole with MVP and at the other pole with p62. B. Impaired recruitment of GFP-LC3 on MVP positive Listeria. HeLa cells were transfected with MVP-tomato (red) and GFP-LC3 (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy, and stained with phalloidin (blue). Inset regions are magnified. The scale bar represents 1 µm. MVP and/or actin positive bacteria were never recognized by GFP-LC3. Arrows point to bacteria at different steps of the infection process: 1) InlK over-expressing bacterium is totally covered by MVP; 2) bacterium is partially labeled with MVP (at the poles) and actin (at the center); 3) bacterium is completely covered by actin; 4) bacterium is enclosed in an GFP-LC3 positive autophagosome. C. Kinetics of autophagy escape for MVP positive Listeria. Jeg3 cells were transfected with MVP-tomato (red) and GFP-LC3 (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, and prepared for real-time video microscopy. Image series were collected every 5 min for 2 h. Time is indicated along the Y axis. The left panel shows that the GFP-LC3 membranous aggregate detaches from the MVP positive Listeria. The entire image sequence can be viewed as Video S2. The right panel shows that the GFP-LC3 membranous aggregate on MVP positive bacteria does not lead to an autophagosome formation, whereas those bacteria efficiently divided. The entire image sequence can be viewed as Video S3. D. Impaired recruitment of GFP-LC3 and ubiquitin to MVP positive ΔactA Listeria. HeLa cells were transfected with MVP-tomato (red) and GFP-LC3 (green), infected with InlK over-expressing ΔactA (ΔactA+pADc-inlK) for 4 h, fixed for fluorescence light microscopy, and stained with anti-ubiquitin antibody (blue) and DAPI (white). Inset regions are magnified. The scale bar represents 1 µm. E. LC3 levels in infected RAW 267.4 macrophages. Left panel: RAW 267.4 macrophages were infected with L. monocytogenes EGD (WT), ΔactA or ΔactA+InlK for 6 h. Cell total lysates were immunobloted for LC3 and actin. Western blot is representative from 3 independent experiments. Right panel: Quantification of the relative LC3-II level (mean ± SEM) shown in the left panel. Statistical analyses were performed on the results of 3 independent experiments using the Student's t test. P values of <0.05 were considered statistically different.
Figure 2(A) Colony formation ability after transfection of SIRT2 plasmids, AGK2 and SIRT2 siRNA. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For A statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. (B) Transwell assay was performed to evaluate the invasion abilities of HCT116 cells after overexpression or downregulation of SIRT2 or treated with AGK2. Stained cells in the lower chambers were quantified. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. (C) Cell migration ability was assessed by wound healing assay in HCT116 cells. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For figure C, statistical significance was assessed with the two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01. (D) Cell cycle distribution was evaluated flow cytometrically after treatment with SIRT2 plasmids, SIRT2 siRNA or AGK2. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For D, statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. (E) Immunohistochemical analysis of SIRT2 protein in representative colorectal tissue specimens is shown (magnification ×100). Weak cytoplasmic staining for SIRT2 was observed in CRC tissues and metastatic tissues, whereas strong cytoplasmic staining was observed in noncancerous tissues.
Figure 5(A) Transcript mRNA expression of Col1a1 and Fn1 in sham and post-MI hearts. Hearts were obtained at day 7 after surgery. Error bars represent mean ± SEM (n=13-16 for each group). Statistical analysis was performed by two-way ANOVA. Post Hoc test was performed by Tukey test. (B) Protein expression of collagen I, fibronectin and αSMA in the infarcted and sham hearts at day 7 after surgery, as assessed by western blotting. Quantified values of proteins of interest were normalized relative to the tubulin band intensity. Error bars represent mean ± SEM. Statistical analysis was performed by unpaired t-test (2-tailed) for fibronectin and αSMA and non-parametric t-test (2-tailed) for collagen I (fibronectin and collagen I: n=5 per group, αSMA: n=3 and 4 for WT-IA and cfKO-IA, respectively). (C) α-smooth muscle actin-positive myofibroblasts (red) were stained in histology sections from infarcted hearts of WT and cfKO mice. Samples were harvested at day 7 after MI. Counter staining was performed by hematoxylin. Scale bar indicates 100μm for top and middle images, 50µm for bottom images. Error bars represent mean ± SEM. Statistical analysis was performed by Mann-Whitney test (n=16 for WT and n=14 for cfKO). (D) Representative images of Picro Sirius Red staining of infarcted heart at 7 days after MI surgery. Images were taken by standard light (top) and polarized light (bottom) microscopes. Scale bar indicates 100μm. Polarized collagen fibers were sorted to orange-red (middle) and green-yellow (bottom) fibers by Image J software. Error bars represent mean ± SEM. Statistical analysis was performed by unpaired t-test (2-tailed) (n=11 for WT and n=13 for cfKO).
miR-155 induces autophagy in macrophages.Figure 4(A-D) RAW264.7 cells were transfected with miR-155 mimic (A and C) or inhibitor (B and D) for 24 h and then either left uninfected or infected with BCG. Expression levels of miR-155 were detected by real-time PCR (A and B). (A-D) RAW264.7 cells were transfected with miR-155 mimic (A and C) or inhibitor (B and D) for 24 h and then either left uninfected or infected with BCG. Expression levels of miR-155 were detected by real-time PCR (A and B). The LC3 levels were detected by Western-blot, and the ratios of LC3-II/β-actin were calculated as shown below the representative blot (C and D) (E and F) RAW264.7 cells transfected with miR-155 mimic (E) or inhibitor (F) was incubated with DMSO or bafilomycin A1 (BafA.) at a concentration of 100 nM for 2 h, and then LC3 levels were detected by Western-blot. The ratios of LC3-II/β-actin were calculated as shown below the representative blot. ***, p<0.001.
Figure 4A Western blot analysis of LC3 and the apoptosis markers phosphorylated p53, p53, and LAMP1 in HC platelets. Platelets were treated with high glucose (25mM), with or without CCCP (10µM). This Western blot is representative of three independent experiments.B Quantification analysis of phosphorylated p53 and LAMP1 (HG; *p=0.018 vs DMSO group, HG/CCCP; **p=0.001 vs HG group, n=4 for each group). C The recognized autophagy inhibitor (2mM 3-Methyadenine; 3MA for 1h) was used to treat DM platelets compared to HC assessing for phosphorylated p53, p53, and LC3I/IID Quantification analysis of phosphorylated p53/p53 was performed. Each value indicated the average band density from a total of three independent samples (DM/3MA; *p=0.026 vs. DM group). E Western blot analysis of PLPs transfected with control or ATG3 siRNA, with or without treatment of 1 mM H2O2. Each lane displayed a different transfected group.F Quantification analysis of ATG3, phosphorylated p53 (ser15), p53, and GAPDH in each group. (ATG3 in ATG3 siRNA; **p=0.0003 vs control group, pp53 in ATG3 siRNA/ H2O2; *p=0.0145 vs. H2O2 group).
Figure 3(A) Plot showing results of the turbidimetric assay (OD 300 nm) conducted to evaluate alginate lyase activity present in the culture supernatants (SN) of the indicated strains carrying the alginate lyase A1-III coding sequence fused to different secretion signals. Culture supernatants (n=1) were collected at 0 h, 24 h or 48 h post-inoculation as indicated. (B) Comparison of the turbidimetric assay results obtained with mpn142 and mpn142Opt secretion signals. Culture supernatants (n=1) were collected at 0 h, 5 h or 24 h post-inoculation as indicated. WT strain and medium (Hayflick) were added as controls.
Sustained H3K4me3 and H3K4me2 at HS memory-related loci depends on HSFA2.Figure 3H3K4me3 (A) or H3K4me2 (B) levels after an acclimatizing HS (ACC) in Col-0 and hsfa2 at HSP18.2, HSP22.0, HSP70 and APX2 as detected by ChIP-qPCR. Col-0 (blue bars) and hsfa2 (orange bars) seedlings were subjected to ACC or no treatment (NHS) 4 d after germination. At the indicated time points after the treatment, ChIP-qPCR was performed with antibodies against H3K4me3 (A), H3K4me2 (B) and H3 (for normalization). Schematics show positions of regions analyzed (grey bars, UTR; black bar, exons). Intergenic control region 1 is 3123 bp (APX2) or 2570 bp (HSP22.0) upstream of the TSS, or 5311 bp (HSP18.2) or 6725 bp (HSP70) downstream of the TSS, respectively. Data are averages over four biological replicates. Amplification values were normalized to input and H3 and the Col-0 4h NHS region 2 (HSP18.2, HSP22.0 and HSP70) or region 3 (for APX2). *, p<0.05; **, p<0.01 for differences between genotypes at the same time point and treatment; squares and triangles within bars mark significant differences (p<0.01 and p<0.05, respectively) between ACC and NHS samples of the same time point and genotype, Student´s t-test. Error bars indicate SE.
Figure 2A In vitro ubiquitin conjugation assay of UbcH7. UbcH7 was incubated with PknG, Ub, and ATP/Mg2+ at 37°C for 30 min. Reaction products were analyzed on a 12% SDS-PAGE gel and stained with Coomassie Brilliant Blue. B In vitro ubiquitin conjugation assay of UbcH7 catalyzed by PknG over time. The reaction products were analyzed as in A. C The ADP/ATP ratios in the ubiquitination reaction mixtures. The reaction mixtures containing 2 μM PknG K181M and 5 μM ATP were incubated at 37°C for 30 min in the absence (reaction 1) or presence (reaction 2) of 5 μM Ub and 5 μM UbcH7. LC-MS MultiQuant software was used to calculate the normalized abundance of each compound (including AMP, ADP, and ATP) by measuring the peak area intensity. D In vitro ubiquitin conjugation assay of UbcH7 catalyzed by GST-tagged PknG or its mutants at 37°C for 30 min. Reaction products were immunoblotted with antibodies against UbcH7 (top panel), Ub (middle panel), and the GST tag on PknG (bottom panel). E In vitro ubiquitin conjugation assay of UbcH7 catalyzed by PknG or His-UBA1 at 37°C for 30 min. The reaction products were analyzed by immunoblotting with antibodies against Ub (top panel), the His-tag on UBA1 (middle panel), and PknG (bottom panel), or by staining with Coomassie Brilliant Blue (bottom). For reducing conditions, samples were treated with 500 mM β-mercaptoethanol (βME) prior to SDS-PAGE gel analysis. F In vitro ubiquitin conjugation assay of UbcH7 catalyzed by PknG or its E1 activity-dead mutant (PknG P57A). Top, the schematic of Mtb PknG. Red arrow, Pro57 site; Bottom, reaction products were analyzed as in A.
Fig.Figure 6A) Top 5 enriched KEGG pathways of differentially expressed genes (abs(fold change) > 2, p < 0.01, Benjamini-Hochberg adjusted) in PD-1-/- vs. wild type microglia. B) Different expression of inflammasome-related genes (abs(fold change) > 1.5, p < 0.05, Benjamini- Hochberg adjusted) in PD-1-/- vs. wild type microglia. C) Different expression of complement system-related genes (abs(fold change) > 1.5, p < 0.05, Benjamini-Hochberg adjusted) in PD-1-/- vs. wild type microglia.
Figure 7DX neuroblastoma cells have MAM-replete mitochondria that are MOMP competent and sensitive to therapeutic stress. REL tumor cells have reduced MAMs (by number and/or proximity) and are relatively multidrug resistant due to attenuated MOMP (red box). Rare tumors at relapse retain MOMP competence and relative therapy sensitivity (green box). Ca2+ transfer may be reduced but is not essential for the multidrug resistant phenotype. Reduced transfer of sphingolipids such as ceramides (Cer) at MAMs is common in REL cells and contributes to attenuated MOMP via altered Bak/Bax oligomerization in response to stress.
Figure 4(A) Colony morphology and AP activity of Control and Yap1 KD ES cells upon differentiation. Morphology and AP staining pictures were taken 2 days after differentiation. (B) A heatmap showing relative mRNA expression levels of 1,995 genes differentially expressed (> 2-fold) between Yap1 KD ES cells and Control upon 4 days of differentiation. Genes were sorted by the fold changes of gene expression between Yap1 KD ES cells and Control (first column). Corresponding gene expression profiles obtained from differentiating ES cells (dESC) are shown in the second column. (C) Relative average module activities (Core and PRC modules) between Yap1 KD ES cells and Control upon differentiation. Data are represented as mean SEM. (D) Colony morphology and AP activity in Yap1 OE cells. Two different Yap1 OE clones (OE1 and OE2) and pool of Yap1 OE (OE pool) were used. Cell morphology and AP staining pictures were taken 3 weeks after electroporation. (E) A heatmap showing relative mRNA expression levels of 2,137 genes differentially expressed (>2-fold) between Yap1 OE ES cells and control ES cells. Genes were sorted by the fold changes of gene expression between Yap1 OE ES cells and control ES cells (first column) and corresponding gene expression profiles obtained from dESC are shown. (F) Relative average module activities (Core and PRC modules) between Yap1 OE cells and control cells are shown. Data are represented as mean SEM. (G) Genes up-regulated in Yap1 OE cells were tested using David 6.7. Significantly enriched gene ontology (GO) terms (biological functions) are shown. Developmental process-related GO terms are highlighted in red.
Figure 7A The senescence phenotypes of five-week-old vector control, proSAG12::CDF4 and proSAG12::CDF4 & 35S::CAT2 plants, from old to young. Scale bars indicate 1.5 cm. B qPCR analysis of the expression of CDF4 and CAT2 in the leaves Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t test. qPCR analysis of the expression of CAT2 in the leaves Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t test. (D) Measurement of chlorophyll in the leaves The third leaves in the rosette were used. Four independent experiments were conducted. Data are presented as the mean ± SD, n=4. *p<0.05 by Student's t test. (E) H2O2 concentrations in the leaves The third leaves in the rosette were used. Four independent experiments were conducted. Data are presented as the mean ± SD, n=4. *p<0.05 by Student's t test.
Figure 6(A) Representative confocal images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 (red) and dsDNA (green) antibody. (B) Representative STED microscopy images of control THP-1 cells processed for IF analysis with cGAS (green), dsDNA (red), and Lamin B1 (cyan) antibodies. (C) The graph depicts the percentage of cells with micronuclei in control siRNA and IRGM siRNA knockdown THP-1 cells. (D-E) Representative STED microscopy images of Irgm1-/- mouse BMDMs (D) transfected with mCherry-cGAS and immunostained with dsDNA (green) antibody. The white line depicts the periphery of the cells. Scale bar, 3 µm. (E) The graph depicts the percentage of cells with micronuclei in Irgm1+/+ and Irgm1-/- mouse BMDMs. (F) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs. (G) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from control and IRGM siRNA transfected HT-29 cells. (H) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT-29 (Rho0) cells. (I) The qRT-PCR analysis with total RNA isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT-29 (Rho0) cells. (J) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs transfected with DNase I (15 μg, 6hr). (K) The qRT-PCR analysis with RNA isolated from control and IRGM siRNA knockdown THP-1 cells electroporated with DNase I (5 μg, 1hr) as indicated. (L-M) Representative confocal images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with (L) dsRNA (green) and Tom20 (red) antibodies. (M) The graph depicts the percentage of Irgm1+/+ and Irgm1-/- mouse BMDMs with dsRNA aggregates. (N) Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with RIG-I (red) and dsRNA (green) antibodies. Line profile: Co-localization analysis using line intensity profiles. Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with (O) Mda5 (red) and dsRNA (green) antibodies Co-localization analysis using line intensity profiles. Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with (P) Mda5 (red), dsRNA (green) and Tia1 (blue) antibodies (Scale bar, 3 µm). Line profile: Co-localization analysis using line intensity profiles.
Figure 6(A) Western blot analysis of HUVECs treated 24 hours prior with COCO and stimulated with VEGF for up to 60 min. Immunoblots are representative of 4 independent experiments. (B) Densitometric analysis of western blot experiments. * P=0.0457; (n=4). (C) COCO treatment does not alter VEGF receptor expression in HUVECs. Immunoblots are representative of 3 independent experiments. (D) Densitometric analysis of western blot experiments; (n=3).