EMBO/sd-character-level-ner huggingface dataset:
Figure 4(A) Serum samples collected from individual mouse (M 1-5) immunized with F 110-136 peptide were tested for antibody binding against F-p27 (110-136) peptide or F 1-34 peptide (control) in ELISA. (B) A549 cells were infected with RSV (MOI = 0.1) for 16 h, and fixed. Cells were treated with mock control rabbit sera (left panels), or rabbit anti-sera against F (center panels), or against F-p27 (110-136) peptide (right panels), followed by Alexa 594 conjugated anti-Rabbit IgG (red). Nuclei are stained with DAPI (blue). Scale bar = 10 µm. The number of cells positive for RSV-F (middle panel) and RSV-p27 (right panel) upon counting of 200 cells were used to calculate percentage of cells stained for each antibody staining are shown in the 'merge' panel. The experiments were performed twice and variation between the two independent expeiremnts was <6%.
Figure 2A Schematic representation of MARS functions. B Cell growth was determined in the absence and presence of 0.5 mM homocysteine or 0.5 mM methionine in the culture media of NE4C cells transfected with pcDNA3.1 vector or pcDNA3.1-Flag-Bcl-2 plasmids (n=3). 5μg plasmids were transfected in each 1×106 cells. The cell index responds to changes in cell number and cell adhesion. Cleaved caspase 3 (c-caspase3) levels were detected to determine apoptosis levels (right). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. C, D Apoptotic cells were detected by flow cytometry. The apoptotic rates of untreated, MARS (C)-, and MARS2 (D)-over-expressing NE4C cells when cultured in the absence and presence of 20 μM homocysteine were normalized to the untreated NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001. E Cells were treated with different methionine concentrations as indicated. Apoptotic rates were detected by flow cytometry and normalized to those of the untreated cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001. F Apoptotic rates were determined in the absence and presence of 20 μM homocysteine in the culture media of control and MARS/MARS2 knockdown NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001. G Metabolic characterization of untreated and MARS-over-expressing NE4C cells based on pathway analysis of significantly changed metabolites. Columns show significantly enriched pathways, and yellow columns indicate oxidative stress-associated pathways.
Figure 4A. Representative western blot showing the changes of O-GlcNAcylation in primary human renal proximal tubule epithelial (HRPTEpi) cells (originating from fetal or adult kidney) treated with different concentrations (0-10 ng/mL) of Stx2a for 24 h. B. LDH cytotoxicity assay following Stx2a (10 ng/mL) treatment of HRPTEpi (adult) cells for 48 h in the presence or absence OSMI-1 (10 µM, final) (n = 3 biological replicates). The effects of Stx2a in vehicle controls were compared with culture medium prepared from HRPTEpi cells maintained in the absence of Stx2a, and OSMI-1 treatment was compared with that of the vehicle (DMSO) controls in the presence of Stx2a. Data information: For graphs error bar represents mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001. C. ELISAs were used to analyze the inhibitory effect of OSMI-1 on cytokine/chemokine production from HRPTEpi cells (adult) exposed to Stx2a (10 ng/mL) for 48 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) control. Data information: For graphs , error bar represents mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001. Global transcriptome analysis of differentially expressed genes of HRPTEpi cells during Stx2a intoxication with vehicle or 10 µM of OSMI-1, respectively. (D) Venn diagram of overlapping differentially expressed genes (DEGs) in the presence or absence OSMI-1 treatment among Stx2a inducible genes compared to cells cultured without Stx2a. Global transcriptome analysis of differentially expressed genes of HRPTEpi cells during Stx2a intoxication with vehicle or 10 µM of OSMI-1, respectively. (E) Pie-chart showing genes significantly regulated by OSMI-1 in the Stx2a-exposed HRPTEpi cells as categorized by function. Global transcriptome analysis of differentially expressed genes of HRPTEpi cells during Stx2a intoxication with vehicle or 10 µM of OSMI-1, respectively. (F) Heat-maps representing the comparative expression levels for DEGs in the presence or absence of OSMI-1 treatment upon Stx2a intoxication related to the inflammatory response (left) and apoptotic process (right). The numbers within the tables are normalized gene expression level compared to HRPTEpi cells maintained in the absence of Stx2a. Expression values are represented with red (upregulation) or blue (downregulation) color using FPKM values by Cufflinks; the cutoffs used a fold change of at least 1.5 followed by pairwise comparison and Student's t test with a Benjamini and Hochberg correction. The FPKM values were normalized using EdgeR within R and visualized using ExDEGA. Data are the means from two independent replicates.
Figure 2Two top-ranked SplashRNA-designed shRNAmirs targeting human Nurd complex components and a control shRNAmir targeting human CD4 were cloned into both miR-AB and miR-E lentiviral vectors with Venus as reporter. The lentiviruses packaged by these constructs were used for transduction of multiple human cell lines. RNAi efficiency was determined by western blot (WB) analysis of target protein levels in the lysates of the transduced cells. -actin served as loading control. A shERWOOD-designed shRNAmir targeting mouse transcriptional factor Tbx21 and a control shRNAmir targeting mouse CD4 were cloned into both miR-AB and miR-E retroviral vectors with Venus or Ametrine as reporter. The packaged viruses were used for transduction of primary mouse P14 TCR transgenic CD8+ T cells, which were used for evaluation of CD8+ T cells differentiation in vivo as described previously(Wang Representative FACS plots of differentiation marker CD127/KLRG1 staining of the gated P14 CD8+ T cells and the statistical summary of SLEC subsets were shown to indicate CD8+ T cell differentiation. Data are shown as mean ± s.d. and n=5. * indicates significant difference (P<0.01, two-tailed unpaired Student's t-test). Data are from one representative experiment of two biological replicates. The Tbx21 RNAi efficiency mediated by miR-AB vs. miR-E in the transduced in vitro-expanded P14 CD8+ T cells was determined by western blot analysis of T-bet protein level. β-actin served as loading control. The RNAi efficiency of Pten.1524 and Pten.932 shRNAmir mediated by miR-AB vs. miR-E was tested in NIH-3T3 cells in the context of <30% transduction efficiency (GFP as reporter). Representative western blots of Pten protein expression of the FACS-sorted GFP+ cells from two biological replicates were shown. β-actin served as loading control. Three shRNAmirs targeting the GFP reporter of a lentiviral vector in miR-AB or miR-E backbone were transduced into MC38 or 293T cells. The RNAi efficiency was determined by FACS analysis of GFP in the context of <30% transduction efficiency. Data are shown as mean ± s.d. and n=3. * indicates significant difference (P<0.05). NS indicates no significant difference (P>0.05, two-tailed unpaired Student's t-test).
Figure 2Each mitochondrial protein is colored according to (A) its fold change calculated by standard differential expression using the limma package (Ritchie et al, 2015; Phipson et al, 2016); (B) its CNV value (5 proteins with the highest CNV value and 5 proteins with the lowest CNV values are highlighted and annotated in boxes); and (C) the absolute difference between the two values (D) Correlation between standard fold change values (left panel) and CNV values (right panel) of proteins quantified in the total lysate and isolated nuclei of HeLa and RKO cells. Only proteins that are differentially regulated (adj. p<0.05) in isolated nuclei are shown (E) Clustering of nuclear protein fold changes estimated from whole cells and isolated nuclei, and comparison to the CNV values obtained from whole cell data (F to H) Comparison of standard differential expression and CNV approach for the three datasets shown in Figure 1-B:D. Volcano plots based on fold changes and adjusted p-values (limma) obtained for (F) lung vs. liver cells (Geiger et al, 2013) (F to H) Comparison of standard differential expression and CNV approach for the three datasets shown in Figure 1-B:D. Volcano plots based on fold changes and adjusted p-values (limma) obtained fo (G) hepatocytes vs. Kupffer cells (Azimifar et al, 2014 (F to H) Comparison of standard differential expression and CNV approach for the three datasets shown in Figure 1-B:D. Volcano plots based on fold changes and adjusted p-values (limma) obtained fo (H) healthy kidney vs. renal carcinoma cells (Guo et al, 2015). Proteins are colored depending on their significance when using the standard limma approach and the CNV approach (based on the four main compartments, q-value<0.1). A stacked barplot (inset) shows the percentage of unique proteins belonging to each category for three q-value thresholds (0.05, 0.1 and 0.25). Related to Figure EV3 and EV4, and Dataset EV1, EV2, EV3 and EV5
Macroautophagic degradation of peroxisomes after transfer of methanol-grown P.Figure 5Macroautophagic degradation of peroxisomes after transfer of methanol-grown P. pastoris cells to ethanol medium. (a-e) Electron micrographs of intermediates seen during macroautophagy. (a) After 1 h of ethanol adaptation, peroxisomes (P) were often surrounded by autophagosomal membranes, higher magnifications of which are in d and e. (b) After 1 h of ethanol adaptation, the autophagosomal membrane surrounding the peroxisomes appears to have fused with the vacuolar membrane. (c) At 2 h a peroxisome in the form of an autophagic body is seen within the vacuole. Magnified pictures of samples obtained at the (d) 20 min time point showing the nuclear endoplasmic reticulum membrane in close proximity to the membranes surrounding the peroxisomes, and at the (e) 1 h time point demonstrating peroxisomes surrounded by multiple autophagosomal membranes. (f) Kinetics of morphological changes in vacuoles and peroxisomal GFP-SKL. Other conditions are described in Materials and Methods, and are the same as in Fig. 4. (g) Fluorescence images taken after 2 h of ethanol adaptation of wild-type P. pastoris cells.
Working model for Xist-mediated PcG recruitment influence on transcriptional silencing based on the phenotypes of the Xist ΔB+C mutant SPEN and proteins of the m6A RNA methylation machinery interact with the A repeat to initiate X-linked gene silencing; PCGF3/5-PRC1 recruitment via hnRNPK interaction with the B and C repeats is responsible for the accumulation of H2AK119ub and concomitant enrichment of the PRC2-mark H3K27me3 over the entire X chromosome.Figure 5SPEN and proteins of the m6A RNA methylation machinery interact with the A repeat to initiate X-linked gene silencing; PCGF3/5-PRC1 recruitment via hnRNPK interaction with the B and C repeats is responsible for the accumulation of H2AK119ub and concomitant enrichment of the PRC2-mark H3K27me3 over the entire X chromosome. In the absence of B and C repeats, there is no enrichment of PcG marks in intergenic regions, but a slight increase at silencing X-linked genes is seen; this could be caused by passive recruitment induced by gene silencing; nevertheless, recruitment of these marks are necessary to stabilize the initial silencing mediated by the A repeat interactors.
A comparison of the SILAC-derived abundance as a function of time, for several proteins that either show differential behaviour between WT and the mutants (Idh2, Idp1 and Aco2) and proteins that behave similarly between all the genetic backgrounds tested (Hsp78).f4A comparison of the SILAC-derived abundance as a function of time, for several proteins that either show differential behaviour between WT and the mutants (Idh2, Idp1 and Aco2) and proteins that behave similarly between all the genetic backgrounds tested (Hsp78). Shown are mean protein ratios (two replicates for mutants, three replicates for WT), error bars indicating peptide-based relative s.d.
Utrophin CH1 domain structure and F-actin interaction sites.Figure 4A. Surface representation of F-actin ADP, five monomers marked as n series from pointed to barbed end. The utrophin CH1 domain in orange interacts with two adjacent actin monomers thus following the actin helical pattern. The crystal structure of dystrophin/utrophin in gray (1DXX) superimposed with cryoEM utrophin CH1 model, boundary of CH1 is marked by an arrow. B. Closer view of utrophin CH1 model, the yellow, orange and red region depicts ABD1, ABD2 and ABD2` sites respectively. The ABD1 and ABD2 sites are restricted to nth actin monomer, the ADB2` site partially interacts with the neighboring n+2nd actin monomer. C, D & E. Residual level information of key amino acids interacting with actin monomers from ABD1: I22, H29 (C); ABD2: N109 (D) ; E. Residual level information of key amino acids interacting with actin monomers from ABD2` site: V87 (E).
, to regulate BMP4 transcription.Figure 8A. The binding of the small peptide EPC5 with the BMP4 promoter region was detected by ChIP-qPCR in ESCs and PGCs (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA). B. Double-luciferase detection of small peptides that affect BMP4 transcription (Data are shown as mean ± SEM, n =3 independent experiments, ** p < 0.01, **** p < 0.0001, one-way ANOVA). C. Effects of m6A on small peptide expression, assessed by western blot (Up), Image J software was used for gray level analysis(Down) (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA). D and E. Western blot(Up) was used to detect gga-mir-12211 and m6A affect the ability of LncBMP4 to translate EPC5. Image J software was used for gray level analysis(Down) (Data are shown as mean ± SEM, n =3 independent experiments, *** p < 0.001,**** p < 0.0001, one-way ANOVA.).
Deletion of ACH1 or MPC1 Results in Upregulation of the Nucleocytosolic Acs2p Pathway, Causing Histone Hyperacetylation (A and B) Representative immunoblot (A) and densitometric quantification expressed as normalized Acs2p/GAPDH ratios (B) of protein extracts from wild-type (WT) and Δach1 yeast chronologically aged to indicated time points in SC 2% glucose medium (see also ).Figure 4(A and B) Representative immunoblot (A) and densitometric quantification expressed as normalized Acs2p/GAPDH ratios (B) of protein extracts from wild-type (WT) and Δach1yeast chronologically aged to indicated time points in SC 2% glucose medium (see also Figure S4).(C) Representative immunoblot analysis of 3-day-old WT and Δach1 cells similar to (A) using pan-acetyl-lysine antibodies. Crude protein extracts (Input) obtained in the presence of histone deacetylase and sirtuin inhibitors (see Supplemental Experimental Procedures) were subjected to immunoprecipitation (IP) at indicated protein concentrations using pan-acetyl-lysine antibodies to enrich acetylated proteins (Ac-Lys IP). GAPDH served as loading control. (D) Heatmap of protein ratios (Δach1 versus WT, log2 scale) corresponding to indicated genes. Data were obtained from mass spectrometric analyses of acetylated proteins enriched by IP similar to (C, 1 mg/ml protein). Extracts from stable-isotope (SILAC)-labeled cells were mixed prior to IP (prelysate), and mean SILAC-protein ratios after IP were normalized to ratios of prelysates to correct for changes in general protein abundance. Two biological replicates were performed (Rep. 1 and Rep. 2) with a regression coefficient of 0.76 of observed protein ratios. Heatmap represents identified proteins with reproducible 1.5-fold upregulation (red color) or downregulation (green color). (E and F) Representative immunoblots of whole cell acid extracts of wild-type (WT) and Δach1 (E) cells chronologically aged to designated time points. Blots were probed with antibodies against total histone H3 (loading control) or H3 acetylated lysines (K9Ac, K14Ac, K18Ac). Densitometric quantification (F) of relative acetylation was calculated as Ac-K/total H3 ratios normalized to ratios of WT at day 1. Data represent means ± SEM (n = 7-8) (see also Figure S5). (G and H) Survival analyses in SC 1.25% galactose/0.75% glucose medium of wild-type cells ectopically overexpressing ACS2 (ACS2 overexp.) compared to vector control cells (Vector control). Survival (I) was determined by colony-forming capacity (clonogenicity) and cell death (J) assessed by propidium iodide (PI)-positive cells analyzed by flow cytometry. Data represent means ± SEM (n = 4) of a representative aging experiment. (I and J) Autophagic flux determination by vacuolar protease-dependent GFP liberation after ACS2 overexpression compared to empty vector controls (Control) similar to (G) and (H). Representative immunoblots at indicated time points (G) and quantification by densitometry of free-GFP/GFP-Atg8p signal ratios (H) are shown. Data represent means ± SEM (n = 4).
Figure 1A. Genome browser view showing chromatin accessibility (ATAC-seq) and ChIP-seq (p300 and H3K4me3) profiles at stage 9, 10½, 12 and 16 at sox2 gene locus. The number at the left (black line) indicates the Y-axis scale of the profile. ATAC-seq peaks were found at promoter (H3K4me3) and enhancer (p300-bound) regulatory regions. B. Chromatin accessibility and p300 binding at differential ATAC-seq peaks visualized using K-means clustering. C. Box plots showing pair-wise sequential-stage comparisons of fold change in accessibility (ATAC-seq) and corresponding changes in gene expression (RNA-seq dataset) (Owens et al, 2016). The data represents two biological replicates. The central band within the boxplot represents the median (50th percentile), the box represents the range between the first and third quartile (25th-75th percentile), and the whiskers show 1.5 x the interquartile range (IQR).
Figure 5A. Astrocyte-specific Daam2 cKO mice show delays in buried food finding. N=13, 15 mice per genotype. Data is presented as mean ± SEM. Student's t-test was used, *p<0.05. B. Illustration of odor discrimination assay. For habituation, each mouse was habituated in a chamber with identical stimulation (odor A) on both sides for 2 minutes, repeated three times. To test for odor discrimination, a newly introduced odor (odor B) was randomly assigned to one of the sides. C. Total travel distance was measured by tracing the nose and body of each mouse in each habituation session of the odor discrimination assay. Both genotypes show no difference in total travel distance. Total N=11, 13 mice were used for each genotype. Data is presented as mean ± SEM. Student's t-test was used. D. Representative traces of nose position tracking of cHet and cKO mice during the discrimination test period. Habituated (odor A) and the new odor (odor B) were placed in the center of the indicated area (odor A: red circle, odor B: green circle). Nose positions within the circled area were indicated with the corresponding color for each odor. E, F. Preference index was tracked and calculated using MATLAB and Optimouse. One sample t and Wilcoxon tests were used for statistical analysis. Data is presented as mean ± SEM. *** p<0.001. Total N=11, 13 mice were used for each genotype.
Figure 3A. Mechanism of D-amino acid oxidase (DAO) in the production of H2O2. Cell lines stably and inducibly expressing a mitochondrial matrix version of DAO (mtDAO) were generated and tested by immunoblot and immunofluorescence. B. Titration of D-alanine (D-Ala) in mtDAO-containing cells and monitoring by matrix HyPer7 (cell generated with the Flp-In T-REX-system). Solid line represents average of multiple measure cells. For area under the curve (AUC) analyses, means of control curves were subtracted from means of D-Ala-treated cells. In the box plot, the central band is the median. The lower and upper hinges correspond to the first and third quartiles. The upper whisker extends from the hinge to the largest value no further than 1.5 times the inter-quartile range, whereas the lower whisker extends from the hinge to the smallest value no further than 1.5 times the inter-quartile range.The Data information: As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. ***, p ≤ 0.001 C. Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO-expressing HEK293 cells with 2 mM D-Ala (cells generated with the Flp-In T-REX-system). HEK293 cells were grown either with glucose (black curve and data points) or galactose (red curve and data points) as carbon source. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information: As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. ***, p ≤ 0.001
Figure 4A, Hourly distribution of wakefulness (WAKE), slow-wave sleep (SWS) and paradoxical sleep (PS) during a 24-h baseline (BL) recording and a second 24-h starting with 6-h sleep deprivation (SD) (recovery: REC) in Ctrl and Fxr1 overexpressing (Fxr1) mice. Significant Group-by-Hour interactions were found for wakefulness during BL (F23,230 = 1.67, p = 0.046) and REC (F23,230 = 2.83, p = 0.0029), for SWS during BL (F23,230 = 1.66, p = 0.045) and REC (F23,230 = 2.89, p=0.0028), and for PS during REC (F16,160 = 2.54, p=0.0018, Two-way ANOVA Huynh-Feldt corrected, *p<0.05). B, Power spectra for WAKE, SWS, and PS in Ctrl and Fxr1 mice computed between 0.75 and 50 Hz per 0.25-Hz for the full 24-h of BL and REC. C, D, The spectral activity of Fxr1 mice expressed relative to that of Ctrl mice for WAKE, SWS and PS during (C) the 24-h BL and (D) the 24-h REC. A significant difference between groups was found for the frequency band 8 to 11.75 Hz during wakefulness (t = 2.30, p=0.044 Student's T-test *p<0.05). E, Time course of wakefulness spectral activity ratio between low alpha (8.5-10.5 Hz) and low theta (4-6 Hz) during BL and REC in Ctrl and Fxr1 mice. A significant Group-by-Interval interaction was found for REC (F22,220 = 1.96, Two-way ANOVA Huynh-Feldt corrected; *p < 0.05), and also when using BL and REC intervals of the light periods (F16,160 = 3.11, p < 0.01),. F, Time course of SWS relative delta activity during BL and REC in Ctrl and Fxr1 mice. A significant Group-by-Interval interaction was found during REC (F13,130 = 1.89, Two-way ANOVA Huynh-Feldt corrected; *p < 0.05).
An imaging-based siRNA screen for gene knockdowns that reduce nuclear size.Figure 1The screen approach is depicted, and details are available in the Methods. Throughout the study, a premalignant breast cancer cell line was used (MCF-10AT1k.cl2), unless otherwise indicated. Images and data shown in this figure were obtained directly from the screen. Scale bar, 20 µm. Montages of representative DAPI-stained nuclei are shown for gene knockdowns that led to reduced nuclear size. Cell-to-cell variability in the amount of ELYS knockdown likely accounts for apparent nuclear size variability. Ten nuclei per column, organized by maximum nuclear size z-score. Scale bar, 25 µm. Median nuclear size z-scores are plotted for gene knockdowns that led to reduced nuclear size. Data are based on three different siRNA oligo sequences for each gene and two biological replicates. Error bars represent the SEM for biological replicates.