EMBO/sd-character-level-ner huggingface dataset:
Figure 4(A) Box plots show the GLUT1 (SLC2A1) gene expression in different breast tumor subtypes in the Servant dataset of 343 primary breast cancer carcinomas, analyzed using Illumina HumanWG-6_v3 Arrays. The gene expression levels are determined using the R2 platform. In the box plot, error bars are the 95% confidence interval, the bottom and top of the box are the 25th and 75th percentiles, the line inside the box is the 50th percentile (median), and any outliers are shown as open circles. **p < 0.01, by Student's t-test.. (B) Real-Time PCR analysis for GLUT1 and MIG-6 mRNA expression in GFP and MIG-6 knockdown BT549 cells (n=3). (C) Immunoblotting analysis for GLUT1 protein expression in BT549 cells with GFP or MIG-6 knockdown. (D) Confocal image analysis results for membrane-bound GLUT1 in GFP and MIG-6 knockdown BT549 cells are shown in the top panel. The arrow indicates membrane-localized GLUT1. Scale bar, 25 μm. Quantitative analysis results of the percentage of GFP and MIG-6 knockdown BT549 cells expressing membrane-localized GLUT1 are shown in the bottom panel (n=3). (E) Representative flow cytometry images (upper) and quantitative results (lower) of measuring glucose uptake in GFP and MIG-6 knockdown BT549 cells (n=3). Cells were grown in the presence of the fluorescent analog 2-NBDG for various time periods, and glucose uptake was quantified using flow cytometric analysis. The red box indicates the gating for 2-NBDG+ cells and the number indicates the percentage of 2-NBDG+ cells. (F) Immunoblotting analysis for GLUT1 protein expression in GFP and MIG-6 knockdown BT549 cells with or without MG132 treatment.
Figure 3(A) G6PD activity is not regulated by lysine succinylation. Purified Flag-tagged G6PD was incubated with or without succinyl-CoA (1 mM) at 30°C for 15 mins, followed by measurement of G6PD enzyme activity as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test). (B) IDH2 activity is suppressed by lysine succinylation. Purified Flag-tagged IDH2 was incubated with or without succinyl-CoA (1 mM) at 30°C for 15 mins, followed by measurement of IDH2 enzyme activity as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments *p<0.05 (two-tailed unpaired t-test). (C) SIRT5 knockdown increases G6PD glutarylation and IDH2 succinylation, respectively, and decreases both enzyme activity. Flag-G6PD or Flag-IDH2 was ectopically expressed in the indicated stable HEK293T cells. G6PD/IDH2 proteins were purified by immunoprecipitated with Flag beads and eluted with Flag peptide, followed by determination of their lysine modifications and enzyme activity. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test). (D) SIRT5 decreases G6PD glutarylation and IDH2 succinylation, and increases both enzyme activity. Flag-G6PD or Flag-IDH2 was co-overexpressed with HA-SIRT5 in HEK293T cells. G6PD and IDH2 proteins were purified by immunoprecipitation with Flag beads and eluted with Flag peptide, followed by determination of their lysine modifications and enzyme activity. The results are average ± SD of 3 independent experiments *p<0.05, **p<0.01 (two-tailed unpaired t-test). (E) Cartoon representation of porcine mitochondrial IDH2 structure shows isocitrate binding pocket (PDB ID: 1LWD) [33], made by Pymol (www.pymol.org). The protein is colored in light green and isocitrate in rainbow, with Lys 360 and Lys 413 residues in IDH2 being colored in red. (F-G) K413 in IDH2 may be a key succinylation site that is directly targeted by SIRT5. The indicated Flag-tagged IDH2 proteins were overexpressed in HEK293T cells with stable SIRT5 knockdown or SIRT5 co-overexpression. Wild-type and mutant IDH2 proteins were purified by Flag beads and eluted with Flag peptide, followed by measurement of IDH2 enzyme activity. The results are average ± SD of 3 independent experiments *p<0.05, n.s.: not significant (two-tailed unpaired t-test).
Adipocyte EV-induced FAO is increased by obesity, but this process is not dependent on increased protein transfer A) Two human (SKMEL28 and 1205Lu) and a murine (B16BL6) melanoma cell lines were exposed, or not, to the indicated EV from primary murine adipocytes obtained from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD), then FAO was measured (n =5 for B16BL6, SKMEL28 HFD EV and 1205Lu ND EV and n=6 for other conditions).Figure 2Two human (SKMEL28 and 1205Lu) and a murine (B16BL6) melanoma cell lines were exposed, or not, to the indicated EV from primary murine adipocytes obtained from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD), then FAO was measured (n =5 for B16BL6, SKMEL28 HFD EV and 1205Lu ND EV and n=6 for other conditions). Volcano plot of mass spectrometry-based quantitative proteomics results showing relative abundance of proteins in primary murine adipocyte EV from obese mice (HFD), as compared to those from lean mice (ND). The dashed lines indicate cut-off values and points colored in grey indicate proteins that display non-significant fold-change by Welch t-test between both conditions (n=3 for ND, 4 for HFD). Proteins involved in FAO are indicated by yellow dots. Western blot analysis of the indicated FAO enzymes in the EV secreted by primary adipocytes from lean (ND) and obese (HFD) mice (top panel) and from human individuals with varying BMI (normal weight, NW; overweight, OW and obese, OB) (bottom panel). For each blot, extracts from 3 independent batches of murine samples or 3 independent individuals for human samples (1-3) are shown. Flotillin 1 (FLOT1) is used as a loading control. Western blot analysis of the indicated FAO enzymes in melanoma cells treated, or not, with EV from lean (ND) and obese (HFD) mice. Tubulin (TUB) is used as a loading control. RT-qPCR analysis of mRNAs for the indicated genes in 1205Lu cells treated or not with EV secreted by primary adipocytes from lean (ND) and obese (HFD) mice for 48 h. Results are expressed relative to the corresponding value for control cells (n=3). Analysis of FAO levels in 1205Lu cells exposed to 3T3-F442A EV and treated, or not, with cycloheximide (CHX) (n=5).
Figure 1Model of an epigenetically primed inducible locus before and after reactivation of TCR/CD28 signaling. Schematic of alternative pathways of cytokine-induced helper T cell differentiation in giving rise to Th1, Th2 or Th17 subtypes of helper T cells. Schematic of T-blast cell transformation. Purified CD4 T cells were activated with ConA for 40 hrs and maintained in IL-2 or IL-7 for 30 hours as TB. Cells were cultured for a further 16 hours +/- IL-2, IL-7 or Ruxolitinib. Cells were re-stimulated (TB+) with 20 ng/ml PMA and 2 mM calcium Ionophore (PMA/I) for 2 hours. Schematics of the procedures for generating Th1 cells in response to intra-venous infection of bacteria (D) and Th17 cells by oral infection with bacteria (E).
Figure 3Coronal brain sections from 2-4-month-old mice of the indicated genotypes were immunostained with different tau antibodies. (A-I) Sections of hemibrain (A-C), whole hippocampus (D-F), and CA1 region (G-I) immunostained for hTau (HT7). (J-O) Hippocampal sections immunostained with phosphorylation-dependent tau antibodies: PHF1 (p396, 404) (J-L), AT8 (pSer 199, 202, and pThr 205) (M-O). (P-R) Representative photomicrographs of GFAP immunostaining of the CA1 region (P, Q) and quantitation of GFAP immunoreactivity (R).
Figure 2(A) Quantification of ss-eGFP-FKBPF36M trafficking in HeLa(M)-C1 cells transfected for 24 h with pmCherry (mCherry) or pmCherry-BspF (mCherry-BspF). Rapamycin was added to initiate secretory traffic of ss-eGFP-FKBPF36M and its colocalization with calnexin (ER), ERGIC-53 (ERGIC), GM130 (Golgi), p230 (TGN), or secretory vesicles (SV) was scored over a 60 min time course. Data are means ± SD from n=3 independent experiments. Asterisks indicate statistically significant differences between mCherry- and mCherry-BspF-expressing cells as determined by a two-way ANOVA with Sidak's multiple comparisons test (P<0.05). (B) Representative confocal fluorescence micrographs of HeLa cells co-transfected for 24 h to produce GFP-TGN38 and mCherry-BspF and stained for F-actin with AlexaFluor™647-phalloidin. Cells were left untreated or treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 1 µm (insets). (C) Quantification of colocalization between mCherry-BspF and GFP-TGN38 in untreated (-CytoD) and Cytochalasin D-treated (+CytoD) HeLa cells. Regions of Interests (ROI, representative shown as insets in panels B) were randomly selected and a Pearson's correlation coefficient was calculated using NIH Fiji image analysis software and Coloc_2 plug-in. Data are means ± SD from n=3 independent experiments in which 2 ROIs from 10 cells (n=20) were analyzed per experiment. The asterisk indicates a statistically significant difference between treatments as determined by a Mann-Whitney test (P<0.05). (D) Representative Western blot analysis of HeLa cells transfected for 24 h to produce HA-BspF, separated into saponin-, Triton X-100- and SDS-soluble fractions and probed for HA-BspF, Hsp27 (cytosol), Calnexin (membranes) and Lamin A/C (nucleus). (E) Schematic depicting key host proteins that control transport pathways associated with the TGN-RE-plasma membrane compartment. Protein colors depict their compartmentalized functions. (F) Representative confocal fluorescence micrographs of HeLa cells co-transfected for 24 h to produce mCherry-BspF and either GFP-Rab6a, GFP-Rab8a or Arf6-GFP and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). (G) Quantification of localization of GFP-Rab6a, GFP-Rab8a and Arf6-GFP on mCherry-BspF-labelled tubules in transfected HeLa cells. Data are means ± SD from n=3 independent experiments, in which at least 300 individual cells per experiment were analyzed.
Solid phase transfection for delivery of Cas9/guide RNA ribonucleoprotein complexes A) Solid phase transfection of nontargeting (scrambled) or CCNA2 targeting RNP complexes into WT RPE-1TP53 -/- cells.Figure 3A) Solid phase transfection of nontargeting (scrambled) or CCNA2 targeting RNP complexes into WT RPE-1TP53 -/- cells. Cells were fixed after 72 hours and imaged after DNA staining with Hoechst. Scale bar, 50μm. B) Quantification of experiments in Fig 3A. Data derived from 2 independent experiments. P value (scrambled vs CCNA2) < 2e-16, Kolmogorov-Smirnov test. C) Solid phase transfection of nontargeting (scrambled), MKI67 targeting RNP complexes into WT RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Ki67 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. D) Quantification of experiments in Fig 3C. Data derived from 2 independent experiments. P value (scrambled vs MKI67) < 2e-16, Mann-Whitney U test. E) Four cell lines were transfected with RNP complexes with scrambled or POLR2A targeting gRNA. 5 days post transfection, cell viability in each well was measured. Results are from at least 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs POLR2A) < 0.005.
Figure 6(A, Confocal images of Bmm-GFP (green) in 3rd instar larval fat bodies of different genotypes. DAPI (blue) stains nuclei. Scale bar represents 25 µm. (A) Knocking down CSN2 or overexpressing RDH/CG2064 in animals with DGAT1 overexpression (ppl>DGAT1) decreases the level and lipid droplet localization of Bmm-GFP. (B) The GFP intensity along the line across a lipid droplet in (A) was measured by ImageJ. The lipid droplet localization of Bmm-GFP, represented by two peaks, is clearly visible in fat cells from ppl>DGAT1 larvae, but it is lost in fat cells from ppl>DGAT1 larvae with CSN2 RNAi or overexpression of RDH/CG2064. More than 30 lipid droplets of each genotype were measured. One typical image curve is shown for each genotype. (C) The level of Bmm-GFP in 3rd instar larval fat bodies of different genotypes was detected by anti-GFP antibody in a western blot. In ppl>DGAT1 larvae, CSN2 RNAi or overexpression of RDH/CG2064 decreases the level of Bmm. D, Confocal images of Bmm-GFP (green) in 3rd instar larval fat bodies of different genotypes. DAPI (blue) stains nuclei. Scale bar represents 25 µm. (D) CSN2 RNAi decreases the level and lipid droplet localization of Bmm-GFP in fat cells from larvae fed on a 30% high-fat diet. , E Confocal images of Bmm-GFP (green) in 3rd instar larval fat bodies of different genotypes. DAPI (blue) stains nuclei. Scale bar represents 25 µm. (E) The level and lipid droplet localization of Bmm-GFP are similar in fat cells from larvae overexpressing DGAT1 or mRFP (control). F) Confocal images of Bmm-GFP (green) in 3rd instar larval fat bodies of different genotypes. DAPI (blue) stains nuclei. Scale bar represents 25 µm. (F) Overexpression of Plin2 decreases the level and lipid droplet localization of Bmm-GFP in fat cells from larvae with DGAT1 overexpression. (G) GFP intensity along the line across a lipid droplet from (F) was measured by ImageJ. The lipid droplet localization of Bmm-GFP, represented by two peaks, was evident in fat cells from ppl>DGAT1 larvae, but is lost in ppl>DGAT1 larvae with overexpression of Plin2. More than 30 lipid droplets of each genotype were measured. One typical image curve is shown for each genotype. (H) 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. bmm RNAi increases the lipid droplet size in fat cells from larvae with DGAT1 overexpression. Scale bar represents 25 µm. (I) Quantification of the lipid droplet size in (H). Data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body. At least 30 cells were analyzed from each fat body. Error bars represent ± SEM. ***: P < 0.001; NS: non-significant. (J) Relative TAG levels in different genetic backgrounds were measured by glyceride assay kit. For quantification, TAG levels were normalized to protein. Error bars represent ± SEM. **: P < 0.01; ***: P < 0.001.
LC3 and p62 accumulate in VCP-KD and IBMPFD mutant–expressing cells.Figure 2.(A) Lysates from scrambled siRNA or VCP-targeted siRNA-treated U20S cells immunoblotted with antibodies to VCP, LC3, p62, or α-tubulin. Densitometric analysis is graphically represented from four independent experiments. LC3 and p62 levels are normalized to loading control. (B) Fluorescent microscopy images of siRNA scrambled control (Scr) or VCP-targeted siRNA KD cells expressing GFP-LC3. Basal numbers of GFP-LC3 puncta/cell were counted. (C) Lysates from control or myc-tagged VCP-WT, ATPase-inactive VCP-EQ, or one of two IBMPFD mutants, VCP-RH or -AE, expressed for 16 h from stably transfected tetracycline-inducible U20S cells and immunoblotted with antibodies to myc, LC3, p62, or α-tubulin. Densitometric analysis is graphically represented from four independent experiments. LC3 and p62 levels are normalized to loading control. Note the increase in LC3II and p62 in VCP siRNA KD and mutant-expressing cells. (D) Fluorescent microscopy images of tetracycline-inducible U20S cells expressing GFP-LC3 treated with and without Baf for 4 h (DMSO control [left] or Baf+ [right]). Cells are control U20S cells and VCP-WT, -RH, -AE, and -EQ. (E) The number of GFP-LC3 puncta/cell was counted for control U20S and VCP-WT, -EQ, -RH, and -AE with and without Baf for 4 h (DMSO or Baf). Note the increase in basal GFP-LC3 puncta in mutant-expressing cells. (A-C and E) Error bars represent the standard error from four independent experiments. *, P < 0.01; and **, P < 0.001. Bars, 25 µm.
Figure 2Schematic representation of the UPAX strategy for phosphopeptide enrichment and MS/MS analysis. siRNA-mediated knockdown of PHPT1 in HeLa cells (24 h) with reference to GAPDH loading control. NT - non-targeting siRNA control. Representative SAX profile of trypsin digested HeLa lysate (Abs280 nm). Representative SAX profile of trypsin digested HeLa lysate (Abs280 nm). Base peak chromatograms are shown for select SAX fractions following high-resolution LC-MS/MS using an Orbitrap Fusion mass spectrometer: (D) fraction 3 (green); (E) fraction 6 (red); (F) fraction 10 (grey). Peptides were fragmented by HCD, with neutral loss of 98 amu from the precursor ions triggering EThCD (Ferries et al, 2017). Tandem mass spectra were separated according to fragmentation strategy in Proteome Discoverer prior to searching with Mascot. The ptmRS node was used for phosphosite localisation. Analysis was performed on three independent biological replicates for each condition (NT or PHPT1 siRNA).
a, b) MCF-7 cells were cultured for 24 h following siRNA with or without FBS before western analysis (a) and cell-viability assays (b).figf3(a, b) MCF-7 cells were cultured for 24 h following siRNA with or without FBS before western blot analysis (a) and cell-viability assays (b). (c) MCF-7 cells were transfected with vector alone (C1), YFP-p27 (WT), or the siRNA-resistant constructs WT-R and T198A-R 24 h before RNA interference (RNAi). PARP cleavage was determined by western blotting and densitometric scanning. Data represent three independent experiments (mean ± s.d.). (d) Western blots of lysates of MCF-7 cells transfected with siRNA before serum-starvation or treatment with LY294002 (LY, 10 μM) or rapamycin (Rap, 100 nM) for 48 h with or without methylpyruvate (MP, 10 mM). (e) MCF-7 cells treated with or without LY294002 for 48 h following RNAi. p27 siRNA pool-1, siRNA1, 2 and 3; arrows, membrane blebbing; triangles, apoptotic bodies. The scale bar represents 50 μm. (f) Western blots of protein extracts from MCF-7 cultured with (+) or without (−) glucose for 24 h following RNAi. Fr, fragment; percentage PARP cleavage = 100 × PARPFr / (PARP + PARPFr).
FK506 impairs calcineurin-dependent TNF-α responses to A.Figure 4A J774A.1 macrophages were treated with an increasing concentration of FK506 and stimulated with AF SC (MOI = 1), zymosan (50 μg/ml) or LPS (25 ng/ml) overnight. TNF-α was measured in the culture supernatant by ELISA. FK506 significantly reduced TNF-α levels at 5 and 10 ng/ml for AF. A significant reduction in TNF-α was also seen for zymosan, but not LPS. B J774A.1 cells were treated with FK506 (10 ng/ml) and either left non-stimulated (NS) or stimulated with AF SC (MOI=1) for 6 h. TNF-α mRNA levels were quantified from whole-cell RNA extracts by RT-PCR. FK506 significantly reduced TNF-α expression. C J774A.1 macrophages were treated with control (Ctrl.) or calcineurin A (CnA)-targeting siRNA (50 nM) and stimulated with either AF SC (MOI = 1), zymosan (50 μg/ml) or LPS (25 ng/ml) overnight. TNF-α was measured in the culture supernatant by ELISA. CnA siRNA led to significant reductions in both AF- and zymosan-dependent TNF-α levels, but had no significant effect on LPS responses.
The fitness of the WT strain background is linked to the number of differences in KO phenotypes for a given condition.Figure 3Barplots reporting the average number of gain and sick phenotypes that are specific to each strain across all pairwise comparisons. Wild type fitness of each strain relative to S288C across the same conditions as in panel A; each dot represents a specific replicate where colony sizes were measured. Relationship between the wild type fitness relative to S288C and the number of conditionally essential genes relative to S288C; each dot represents a strain-condition replicate as in panel B. The conditions maltose, glycerol, and NaCl are highlighted. Changes in gene deletion phenotypes for growth on osmotic stress conditions. The top heatmap contains genes belonging to the HOG pathway, while the bottom one those genes whose growth phenotypes varies the most between Y55 and YPS. Significant growth phenotypes are marked with "-". Changes in deletion phenotypes for growth on glycerol and maltose. The top heatmap contains the MAL genes, while the bottom one those genes whose growth phenotypes vary the most between S288C and the other three strains. Significant growth phenotypes are marked with "-".
PTX-mediated regulation of local translation.Figure 5(A) Representative pictures of nascent Camk2a-peptides in mock- and PTX-treated neurons. Upper Panels: Representative overview images with merged channels (grey: MAP2-staining, red: Duolink-PLA signal, blue: Hoechst nuclear staining). Middle Panels: Magnifications of boxed areas marked in (A). MAP2 signal was used to outline somata and dendritic processes (white lines). Lower Panels: Negative control of puromycin-untreated cells with representative Duolink-signal in dendrite and soma, respectively. (Scale bars: in overview images 20 μm, in inserts of higher magnification 5 μm). (B) Same as A with nascent Synapsin1-peptides. ( (C) Quantification of Duolink-particle density of Figure 5A and B assessing PTX-treatment effect. n=3 independent experiments with 10 images averaged per condition and experiment; Three-way ANOVA followed by Tukey's post-hoc multiple comparison test; *p < 0.05; **p < 0.01). (D) Quantification of Duolink-particle density of Figure 5A and B assessing Puromycin-treatment effect. n=3 independent experiments for Puromycin-Minus and n=6 datapoints for Puromycin-Plus conditions (PTX- and mock-conditions combined together across n=3 independent experiments, taken into account in ANOVA-model) with 10 images averaged per condition and experiment; Three-way ANOVA followed by Tukey's post-hoc multiple comparison test; **p < 0.01, ***p < 0.001).
Figure 5Macroscopic appearance of adult testes from an 8-week-old WT (Spatc1l+/+), heterozygous (Spatc1l+/-) and homozygous (Spatc1l-/-) mice. Scale bar = 2 mm. Comparison of testis weight in WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) mice. The organs were trimmed of fat and weighed. The average testis weight/body weight ratios for WT, Spatc1l+/-, and Spatc1l-/- males were 0.0034±0.0001, 0.0038±0.0003, and 0.0031±0.0002, respectively. Data are presented as means ± s.d. (n = 7). Macroscopic appearance of the adult epididymis from WT, Spatc1l+/-, and Spatc1l-/- mice. Scale bar = 2 mm. Epididymis weight in WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) mice. The average epididymis weight/body weight ratios for WT, Spatc1l+/-, and Spatc1l-/- males were 0.001±0.0001, 0.001±0.0001, and 0.001±0.00008, respectively. Data are presented as means ± s.d. (n = 7). Histological analysis of hematoxylin and eosin-stained sections of seminiferous tubules prepared from testes of WT and KO mice. Scale bar = 100 μm (upper) and 50 μm (lower). Number of mature epididymal sperm from WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) mice. Sperm were collected from the vas deferens. The average number of mature sperm in WT, Spatc1l+/-, and Spatc1l-/- males was (4.06 ± 0.40) × 106, (3.7 ± 0.7) × 106, and (3.93 ± 0.90) × 106, respectively. Data are presented as means ± s.d. (n = 5). Morphological analyses of epididymal sperm in WT, He and KO mice. Epididymal sperm were observed under an inverted microscope. The arrows indicate separated tails, black arrowheads indicate separated heads and white arrowheads indicate folded heads. Scale bar = 50 μm. Percentage of headless sperm from cauda epididymides of WT (black), Spatc1l+/- (gray), and Spatc1l-/- (white) mice. The average percentage of headless sperm in WT, Spatc1l+/-, and Spatc1l-/- males was 6.3, 52.9 and 100, respectively. Each dot represents percentage of headless sperm in an individual mouse, and medians are represented as horizontal lines. (n=13, *P < 0.001, Student's t-test). Immunostaining analysis of sperm heads and tails in WT and KO mice. Sperm tails were observed by immunostaining for α-tubulin antibody (red), and counterstaining with the nuclear dye Hoechst 33342 (blue). Sperm were also observed by differential interference contrast (DIC) microscopy. Scale bars = 20 μm (upper) and 5 μm (lower).