EMBO/sd-character-level-ner huggingface dataset:
Figure 4(A) Top: Images taken by confocal microscopy showed the localization of LOC113230 detected by RNA scope assays in SW-1116. Scale bar represents 50 μM. Bottom: qRT-PCR assays showed LOC113230 expression level in the nuclear and cytoplasmic fractions extracted from SW-1116 cells. Silver staining images showed ASS1 as a novel LOC113230 interacting protein. The LOC113230 interacting proteins were identified by incubation biotin labeled LOC113230 with cytoplasmic extracts prepared from SW-1116 cells and the co-eluted proteins were identified by Mass spectrometry. NC, Negative control. RIP assays were employed to validate the association of ASS1 with LOC113230 in SW-1116 cells. Antibodies against HuR, GAPDH or control IgG were served as controls. Western blot assays showed that LOC113230 specifically precipitated ASS1 in SW-1116 cells. PC, Positive control. Western blot assays showed level of ASS1 in SW-1116-Vector and SW-1116-LOC113230. Top: western blot assays showed level of ASS1 in SW-1116 cells stably expressing LOC113230 or vector treated with cycloheximide (CHX, 20 μg/mL) or vehicle for the indicated periods of time. Bottom: the gray scale-analysis of ASS1 level was conducted and GAPDH protein level was used as a loading control. Results are shown as means±SD. SW-1116 cells stably expressing LOC113230 or vector were treated with MG132 (5 μM) or vehicle for 4, 8 or 12 hrs, respectively. Cell lysates were analyzed by western blotting assays. SW-1116 cells bearing stable expression or knockdown of LOC113230 were treated with MG132 (5 μM) for 12 hrs. Cell lysates were immunoprecipitated (IP) assays were performed with α-Flag antibody. The co-eluted proteins were detected by western blot assays with ubiquitin (Ub), ASS1 and α-Flag antibodies, respectively. Tandem mass spectrometry analysis identified the ubiquitinated K234 residue of ASS1 in the presence of LOC113230 expression. The protein level of ASS1 was examined by immunoblotting in SW-1116-Vector and SW-1116-LOC113230 cells overexpressing ASS1WT or ASS1K234R . SW-1116 cells stably expressing ASS1WT, or ASS1 K234R and LOC113230 or not were treated with MG132 (5 μM) for 12 hrs. Cell lysates were immunoprecipitated (IP) assays were performed with α-Flag antibody. The co-eluted proteins were detected by western blot assays with ubiquitin (Ub) and α-Flag antibodies, respectively.
Figure 1A. Purified trophozoite-stage iRBCs and matched uRBCs were labeled with one of ten 13C-substrates (listed in purple). All mass-to-charge (m/z) features identified by LC-MS were extracted and isotopologues grouped. 13C-labelled and 12C-labelled isotopologue groups were compared to identify m/z features in each cell type and that were differentially labelled between iRBC and uRBC. Putative metabolite identities were then confirmed with authentic standards, MS/MS spectral matching, 13C-labelling profile, and exact mass. The observed metabolic network of each cell type was then constructed using the draft metabolome and 13C-labelling information and compared to the predicted P. falciparum metabolome reported by Huthmacher et al. (2010). B. All m/z features detected from iRBC extracts plotted as the log2 ratio of their abundance detected in 13C-glucose versus 12C-glucose conditions. 859 m/z features (from a total 33,691 m/z features identified) were significantly altered in 13C versus 12C samples of iRBC. Highlighted in black is a single m/z feature (m/z 275.0167). C. The 859 significant m/z features (x-axis; putative M0 species) were ranked by their fractional enrichment (y-axis) and the observed mass queried against the METLIN metabolite database. m/z features that returned a hit were classified as presumptive features (blue) and retained for validation. Presumptive m/z features displayed a wide range of fractional enrichments, indicating no systematic bias. D. Changes in the isotopologue group distribution in 12C-glucose and 13C-glucose labelled samples of m/z 275.0167. Data are presented as the mean 13C-fractional enrichment ± SEM from six biological replicates. E. MS/MS fragmentation of m/z 275.0167 matched the reference spectrum of 6-phosphogluconate, consistent with the exact mass and labelling pattern, confirming the metabolite identity. This approach was repeated for all presumptive m/z features.
Figure 1A slice through a filtered tomogram showing SR vesicles in contact with a putative T-tubule. Individual RyR1 molecules are indicated by the red circles. The dense protein coat on the surface of the SR vesicles correspond to the SERCA pump. Areas circled in yellow indicate accumulation of density inside the SR. Scale bar: 20 nm. A slice through the structure of RyR1 in contact with putative TT-membrane. A middle slice through the in situ structure of RyR1 at 12.6-Å resolution. The observed additional transmembrane density is indicated by the red arrows. Scale bars in B, C: 10 nm. Slice through the structures of apoRyR1 with the putative TT-membrane (left) and the standalone apoRyR1 (right, with the flipped handendness and reduced resolution for comparison) 22 Å away from the middle slice (Y = 92 in the volume coordinates). Scale bar: 10 nm. Volume-rendered visualizations of the average structure of RyR1 fit with the RyR1 atomic model (PDB: 5TB2). The left panel is a thin section through the center of the volume. The location of the CaM/S100A1 binding site is marked with red circles in slices through maps of the in situ structure (left) and the single particle structure (EMDB: 8393) in the same orientation as the in situ structure and filtered to 15 Å (right). Scale bar: 10 nm. The in situ structure with an atomic model (PDB: 5TB2) fitted showing an unaccommodated density. Red circles are sites of potential interaction between RyR1 and the regulatory proteins.
Figure 1A Family pedigree. Blackened symbols indicate the patients with hemophilia B; white circles with dots show carriers of the mutation; open symbols indicate healthy individuals. Circles represent females; squares represent males. The proband is labeled with an arrow. B The partial sequences of the F9 gene from a healthy subject (left), the patient (middle) and a carrier (right). C Amino acid alignment of partial sequence of the serine protease domain of FIX in 10 species. An asterisk (*) indicates positions which have a single, fully conserved residue. A colon (:) indicates conservation between groups of strongly similar properties. A period (.) indicates conservation between groups of weakly similar properties.
Figure 2(a) Top: Scheme of the IgA-switching assay in CH12F3-2A cells. After electroporation of siRNAs, cells were cultured for 24 h, and then stimulated by CIT, cultured for another 24 h, and subjected to FACS analysis. Bottom: Confirmation of the siRNA-mediated KD of SAMHD1 in CH12F3-2A cells. (b) FACS profile of the percentage of CH12F3-2A cells undergoing IgA switching after transfection of the indicated siRNAs into CH12F3-2A cells cultured with (+) or without (-) CIT stimulation for 24 h. The number in the FACS plot represents the percentage of cells expressing IgA on their surface. (c) Summary of the FACS data obtained from three independent experiments. Data are shown as the % IgA switching relative to the IgA switching in cells transfected with control siRNA (siCONT) (n=3; mean + sd; two tailed unpaired Student's t-test). (d) Quantitative RT-PCR analysis of the μGLT, αGLT, Aid, and Samhd1 expression in the indicated samples. The data were normalized to Gapdh, and represents as the mean of the two independent experiments with standard deviations. Statistical significance as determined by two-tailed unpaired Student's t-test is shown. ns = not significant. (e) Confirmation of the CRISPR/Cas9 mediated KO of Samhd1 in CH12F3-2A cells. Beta-actin was used as a loading control. (f) CSR time course analysis of WT and Samhd1 KO clones. The data are shown as percentage of IgA expressing cells (n=2; mean ± sd; two tailed unpaired Student's t-test; **, p<0.01). (g) Proliferation of WT and Samhd1 KO clones after stimulation with CIT (n = 2; mean ± sd; two tailed unpaired Student's t-test; ns = not significant). (h) Analysis of the cell proliferation of WT or Samhd1 KO clones of CH12F3-2A. Histograms show the CFSE dye dilution derived from the indicated samples. Aphidicolin (2 μg/mL) was also used as a positive control. (i) Schematic representation of the IgG1 or IgG3 switching assay in spleen B cells. Bottom: Western blot analysis of the SAMHD1 expression in spleen B cells derived from WT or Samhd1 KO mice. Tubulin was used as a loading control. (j) Bar graphs show the compilation of IgG1 and IgG3 CSR assays from five independent experiments. The data are shown as percentage of IgG1 (left) or IgG3 (right) expressing cells (n=5; mean ± sd; two-tailed unpaired Student's t-test).
Lipid droplets are found in membrane protrusions, at proximity to mitochondria, in melanoma cells exposed to adipocyte EV A) Left panel, indicated melanoma cells exposed to 3T3-F442A EV were fixed, stained with BODIPY and Phalloidin before observation by confocal microscopy.Figure 6Left panel, indicated melanoma cells exposed to 3T3-F442A EV were fixed, stained with BODIPY and Phalloidin before observation by confocal microscopy. Right panel, quantification of the percentage of cells presenting lipid droplets (LD) within membrane protrusions (n=6). Left panel, melanoma cells exposed to EV secreted by adipocytes from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) were fixed, stained with BODIPY and Phalloidin before observation by confocal microscopy. Right panel, quantification of the percentage of cells presenting lipid droplets (LD) within membrane protrusions (n=5 for SKMEL28 and n=6 for 1205Lu). 1205Lu cells exposed to 3T3-F442A EV were stained with a MitoTracker probe. Then, cells were fixed and stained with BODIPY before observation by confocal microscopy. A zoomed crop of the area containing mitochondria and lipid droplets in a membrane protrusion is shown (scale bar: 5µm). Transmission electron microscope observations of 1205Lu cells exposed, or not, to 3T3-F442A EV. Mitochondria are colored in pink and lipid droplets are colored in yellow on images on the right. Number of lipid droplets (LD) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed, or not, to 3T3-F442A EV (3T3-EV) (n=28 for control and n=27 for 3T3-EV). Area of lipid droplets (LD) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed, or not, to 3T3-F442A EV (n=19 for control and n=25 for 3T3-EV). Left panel, 1205Lu melanoma cells were exposed, or not, to 3T3-F442A EV. Then, live cells were stained with LysoTracker and BODIPY probes and observed by confocal microscopy. Right panel, quantification of the percentage of cells presenting lysosome within membrane protrusions (n=3).
Figure 4A UV-visible absorption spectra of recombinant DJA6 and DJA5. Recombinant DJA6 after elution from Ni-NTA Sepharose beads followed by concentration is shown on the right. B Electron paramagnetic resonance spectra of recombinant DJAs. The observed resonance is marked for the g value. C Analysis of the ferrous iron binding activity of DJA6. Isothermal titration calorimetry measurements of ferrous iron binding to apo DJA6 were carried out on a MicroCal iTC200. Fe2+ was titrated into a solution of 50 μM DJA6 or DJA6 variants. For the background titrations, the ferrous iron was titrated into the corresponding buffer. D DJA6 can bind Fe2+ in chloroplast. Arabidopsis mesophyll protoplasts were transformed with plasmids that express the indicated gene constructs, respectively. The untransformed protoplasts and protoplasts transformed with non-fused EBFP proteins were used as controls. The protoplasts were further stained by FeRhoNoxTM-1 (red) to visualize the cellular distribution of Fe2+. Green, EBFP fluorescence signals; magenta, chloroplast autofluorescence signals; yellow, regions where the EBFP and FeRhoNoxTM-1 fluorescence signals merged. The speckles which DJA6-EBFP co-localizes with FeRhoNoxTM-1 are indicated by white arrows. Scale bars = 5 μm. E Diagram of the domain structure of DJA6. Grey, TP (chloroplast transit peptide); blue, J domain; steel blue, GR domain; cyan, CR domain; green, C-terminal domain. F Ribbon representation of the quarternary structural model of mature dimeric DJA6 prepared with PyMOL (Lam & Siu, 2017). The α subunit and α' subunit of DJA6 dimer are colored blue-to-green and green-to-red, respectively. The relative orientations of the domains differ in the two monomers in the asymmetric unit. G UV-visible absorption spectra of recombinant DJA6 and DJA6 truncated peptides. Recombinant DJA6 and DJA6 truncated peptides after elution from Ni-NTA Sepharose beads followed by concentration are shown above. H Size-exclusion profiles for apo and iron-loaded forms of DJA6. 1 mM EDTA, purified DJA6 after incubated with EDTA at a final concentration of 1 mM; 20 μM or 100 μM Ferrous, apo-DJA6 after incubated with Fe(NH4)2(SO4)2 at a final concentration of 20 μM or 100 μM, respectively. Positions of theoretical tetramer and dimer are indicated by dotted lines. Images of the Coomassie Brilliant Blue-stained gel after SEC elution are shown.
The viability of atg mutants in buffered starvation medium.Figure 1(A) WT and atg1Δ cells grown in YEPD medium were transferred to SD-N or SD-N +50 mM MES-KOH (pH 6.2) medium for 120 hours, and dead cells stained by phloxine B were observed by fluorescent microscopy. Scale bar, 25 µm. (B-C) WT and atg1Δ cells grown nitrogen starved as (A) for the indicated times. Cell viability (B) and medium pH (C) were examined by phloxine B staining and pH meter, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.
Figure 7(A) SMAD3 signature has been established by comparing BRAFi-resistance genes and SMAD3-regulated genes identified by chromatin immunoprecipitation followed by DNA sequencing (ChIP-Seq) (B) Basal SMAD3-signature in six melanoma cell lines (S for sensitive to BRAFi and R for Resistant). (C) Inducibility of SMAD3-signature in six melanoma cell lines exposed to TGF-β (10 ng/mL, 48h). Data were normalized to cell lines exposed to solvent (4mM HCl + 1mg/mL human BSA). Values obtained with the TGF-β stimulated M238R cell line have been set to 1. (D) SMAD3-signature discriminates differentiation states of 53 melanoma cell lines The SMAD3-signature in four subgroups of melanoma cell lines. Each point represents a cell line (n= 17, 12, 14 and 10 respectively for melanocytic, transitory, neural crest-like and undifferentiated cell lines); Each histogram represents the median with interquartile range (E) Heatmap depicting mRNA levels of SMAD3-signature in BRAF(V600E) non-treated melanoma patients (dataset from TCGA; SKCM, BRAF(V600E) mutated: n=118). Three pigmentation genes (MITF, MLANA and TYR) have been added to highlight the differentiation states of tumors. ~20% of tumors are considered as dedifferentiated tumors (red box) with a high SMAD3-signature. (F) SMAD3-signature in pre-treatment biopsies from responders and non-responders to anti-PD-1 treatment (from the Hugo's cutaneous melanoma cohort, n=15 and 13 respectively) (Hugo et al, 2016). (G) SMAD3-signature in two groups (before BRAFi treatment or during relapse) of V600E patients (14 on 16 patients displayed an upregulation of SMAD3- signature during relapse). (H) SMAD3 depletion decreases expression of SMAD3-regulated genes. SMAD3 knock-down by siRNA decreased mRNA expression of BRAFi-resistance genes in SKMel28R and Me1402. NRP1 mRNA was not detected in our experimental conditions. (I) SMAD3 signature overlapped with mesenchymal signature in melanoma tumors (TCGA, n=459) Melanocytic signature highlights the differentiation states of tumors SMAD3 signature and mesenchymal signature correlate in melanoma tumors (TCGA, n=459) (J) The SMAD3 signature overlaps with the glioblastoma mesenchymal subtype
UVRAG inhibits mitochondrial translocation of BAX.Figure 5 UVRAG inhibits mitochondrial translocation of BAX. After transfection with indicated shRNA or cDNA for 48 h, HL60 cells were treated with doxorubicin (Doxo) and UV irradiation for 12 h, and then BAX in cytosol (Cyt) and mitochondria (Mit) was assayed by western blotting (A-C) and activation of BAX (D-F) was assayed by western blotting after IP using BAX monoclonal antibody (clone 6A7) *P0.05. AU, arbitrary units; BAX, BCL2‐associated X protein; CHAPS, 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulphonate buffer; Cyt c, cytochrome c; IP, immunoprecipitation; shRNA, short‐hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.
a) Transcriptional levels of hlh-30 and the mxl-3-dependent lipl genes measured in 5 h fasted wild-type (WT) or hlh-30(tm1978) young-adult worms depicted as mean ddCt+s.figf4(a) Transcriptional levels of hlh-30 and the mxl-3-dependent lipl genes measured in 5 h fasted wild-type (WT) or hlh-30(tm1978) young-adult worms depicted as mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms show that hlh-30 is induced following fasting and HLH-30 induces lysosomal lipolysis. N = 4 independent experiments for lipl data, n = 2 independent experiments for hlh-30 mRNA. HLH-30 deficiency fully abrogates lipl-2, 3 and 5 (P0.0001), and impairs lipl-1 (P0.01) transcriptional activation following fasting. (b) Animals treated from late L3 stage with RNAi against TOR or vector control were harvested as young adults. Mean+s.e.m. of three independent experiments shows that inhibition of TOR is sufficient to induce hlh-30 transcription. (c) L3 animals carrying the rescuing construct hlh-30P::HLH-30::mGFP::hlh-30 3′UTR (mGFP, monomeric GFP, and UTR, untranslated region), well fed or fasted for 8 h show that HLH-30 is enriched in intestinal nuclei of fasted worms (exposure time: well fed, 500 ms; fasted, 100 ms). (d) ORO staining of wild-type or hlh-30(tm1978) young adults fasted for 8 h reveals that hlh-30 is required for optimal lipid mobilization on fasting. Mean percentages+s.e.m. are shown below relative to well-fed worms treated in parallel, n = 3 independent experiments. (e) mxl-3 and hlh-30 transcriptional levels in wild-type, hlh-30(tm1978) and mxl-3(ok1947) mutants in the basal and fasted states show that mxl-3 transcription is hlh-30 independent, and hlh-30 transcription is mxl-3 independent. Expression is presented relative to wild-type fed animals as mean ddCt+s.e.m. No significant differences relative to wild type in the same feeding state were observed, n = 3 independent experiments. (f) Representative ChIP-qPCR analysis of well-fed and 6 h fasted mixed stage worms expressing HLH-30::GFP presented as in Fig. 3d shows that HLH-30 occupies the lipl promoters during early fasting. See raw data of two independent experiments in Supplementary Table S9. (g) Transcriptional levels of the mxl-3-dependent lipl genes in wild-type, mxl-3(ok1947), hlh-30(tm1978) or mxl-3(ok1947);hlh-30(tm1978) double mutant young-adult worms show that hlh-30 suppresses the constitutive-induction-of-the-lipl-genes phenotype of mxl-3 mutant animals (mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms). n = 3 independent experiments.
Pol λ discriminates against extension past an 8-oxo-dG(syn):dA mispair.Figure 7(A) Glu529 (purple) forms a hydrogen bond with the C2-amino group of the primer terminal templating 8-oxo-dG (orange) in the anti-conformation. (B) This interaction is abolished when 8-oxo-dG (orange) adopts the syn-conformation. An overlay with the 8-oxo-dG(anti)-containing structure reveals that Glu529 (black) would likely clash (red-dotted line) with the C8-carbonyl of 8-oxo-dG(syn). Repositioning of Glu529 (purple) places the side chain 4.5 Å away from the C8-carbonyl. (C) Comparison of catalytic efficiencies for extension past an 8-oxo-dG(anti):dC, 8-oxo-dG(syn):dA and dG:dC base pairs at the primer terminus using WT and E529A Pol λ.
Figure 1(A) Schematic illustration of the screening strategy. (B) Quantification of embryos that expressing the ZGA reporter (MERVL::tdTomato) after injection with a siRNA-repressor or mRNA-inducer. N, the total number of embryos analyzed for each condition. Error bars, mean ± S.D.; n = 3 biological replicates per group. (C) Representative fluorescence image of SCNT embryos derived by different methods. The SCNT embryos produced by transfer of MERVL::tdTomato cumulus cells (B6D2F1 background) into WT enucleated oocytes. n = 3 biological replicates per group. Scale bar, 100 μm. (D) Stacked bar plots show the fraction of embryos at blastocyst stages after injection with different mRNA, as indicated. Error bars, mean ± S.D.; n = 3 biological replicates per group; N, total number of embryos analyzed for each condition; **P < 0.01, ***P < 0.001 as compared with control group, by two-tailed Student's  t-test. (E) Representative live-cell images of dynamic ZGA-reporter expression during SCNT embryo development. The time after initial observation is shown at the bottom of the image. n = 3 biological replicates per group. Scale bar, 50 μm. (F) Schematic transient induction of Dux expression. All timings reported in this paper is hour post-activation. (G) Preimplantation development of SCNT embryos. The percentage of embryos reaching each indicated stage is shown. The D-SCNT embryos produced by transfer of dox-Dux cumulus cell (B6D2F1 background) into WT enucleated oocytes (B6D2F1 background). Error bars, mean ± S.D.; n = 3 biological replicates per group. Scale bar, 100 μm. (H) Unsupervised hierarchical clustering. (I) Venn diagram showing the overlap between the genes that failed to be activated in SCNT 2-cell embryos and derepressed in D-SCNT. Heatmap, GO terms showing the expression pattern of 257 overlapping genes. (J) Image of full-term cloned mice derived by canonical SCNT and D-SCNT method (left). The birth rate of SCNT embryos derived by different methods is indicated (right). N, the total number of cloned pups obtained from each condition.
Tet1 upregulation is a mediator of Kdm3b function during reprogramming a.Figure 2Pearson correlation of transcripts per million (TPM) of WT and Kdm3b-KO pre-iPSC upon DMSO or AA+2i treatment for 2 or 10 days and mESC, reprogramming populations from Chronis et. al (2017), (labelled as CC). k-means clustering of the union of DEG (FDR > 0.99) between day 2 and DMSO in WT and Kdm3b-KO pre-iPSCs. Values represent a log2-fold change of day 2 over DMSO. Key pluripotency genes are shown on the right. k-means clustering of the union of DEG (FDR > 0.99) between day 10 and DMSO in WT and Kdm3b-KO pre-iPSCs. Values represent a log2-fold change of day 10 over DMSO. Key pluripotency genes or epigenetic regulators are shown on the right. Expression of Tet enzymes and Tdg during AA+2i treatment in WT or Kdm3b-KO pre-iPSCs. Error bars represent the standard deviation of two biological replicates from the RNA-Seq sample. Quantification of percent Nanog-GFP cells of WT, Kdm3b-KO and Kdm3b-KO pre-iPSC clones expressing Tet1 catalytic domain (Tet1CD), Nanog, or both.
Figure 5A) Manhattan plot of genome-wide association for TAG(48:2). Red line shows Bonferroni-corrected threshold of significance calculated based on FaST-LMM pvalues. B) Locuszoom plots showing the focused genomic region (x-axis) plotted against the -log10 (p-value) of association for liver mRNA expression of Map2k6. P-values for GWAS associations were calculated using FaST-LMM (C) Correlations between hepatic expression of Map2k6 and all TAGs identified in the study. Blue represents negative correlations. D) Experimental design for validation of Map2k6 as a regulator of hepatic TAGs and phenotypic traits, which changed as a result. E) Western blots of liver homogenate using anti-Map2k6 and anti-β-actin. F) Comparison of total hepatic TAG between Map2k6-overexpressing mice (black bar) and control mice (empty bar). G) Differences in total phospholipid (PC), total cholesterol (TC) and unesterified cholesterol (UC) between Map2k6-overexpressing mice (black bars) and control mice (empty bars). H) Body fat % of both experimental cohorts prior to (week 0) or 8 weeks on a HF/HS diet. I,J) Plasma concentration of glucose (I) and insulin (J) at the end of the 8 weeks study.