EMBO/sd-character-level-ner huggingface dataset:
ROS generation by STAMP2 depends on its ferrireductase activity.Figure 8A 293T cells were transfected with plasmids expressing either an empty vector (Vec) or vectors expressing HA-tagged STEAP, STAMP1 (ST1), STAMP2 (ST2), or STAMP3 (ST3). Whole-cell extracts were prepared and subjected to Western blot analysis using anti-HA antibody or GAPDH as a loading control. B Relative ferrireductase activities were measured in the cells expressing the different constructs used in (A) as described in Materials and Methods. *P < 0.0001. C 293T cells were transfected with different plasmids expressing either an empty vector (Vec) or vectors expressing His-tagged wild-type STAMP2 (WT) or three STAMP2 mutants (dGSR, H304L, or H397L) as indicated. Whole-cell extracts were prepared and used in Western blot analysis with anti-His antibody or GAPDH antibody as a loading control. D Relative ferrireductase activities were measured in the cells from (C) as described in Materials and Methods. *P = 0.0003. E 293T cell line with Dox-inducible STAMP2 expression or vector control was generated. Western blot analysis confirmed STAMP2 expression in a Dox-inducible manner. F The cells from (E) were either left untreated or treated with increasing amounts of Dox, and ferrireductase activity was determined. *P < 0.0001. G The cells with Dox-inducible STAMP2 expression from (E) were either left untreated or treated with Dox for 48 h. Prior to ferrireductase activity measurement, the cells were treated with or without DPI as indicated for 1 h. *P < 0.0001; **P = 0.0012; ***P = 0.048. H The cells from (E) were treated with 100 ng/ml Dox for 2 days. Oxidative stress was then measured by NBT staining, and the stained cells were photographed. There were equal numbers of cells on the plates for −/+ Dox as shown in Supplementary Fig S4. I The cells with Dox-inducible STAMP2 expression from (E) were either left untreated or treated with Dox for 48 h. Then, the cells were treated with or without 10 μM DPI before being subjected to NBT staining. *P < 0.0001.
Expression analysis of TEs.Figure 4(a) Box plot summarizing differential expression of unambiguously mapped individual TEs, compared with all protein-coding genes, or housekeeping genes. Box plots outline the interquartile range (IQR) with the median and whiskers +/- 1.5 IQR . For all p-values, see also Appendix Table EV6. ** indicate a p-value smaller than 1.01e-11 (two sided non paired Wilcoxon rank sum test). (b) Heatmap of expression differences for all 318 Arabidopsis TE families in stem cells relative to non-stem cells. (c) Number of TE families with at least 2x expression difference. E = nuclei from embryos, D7/14/35 = nuclei from 7/14/35 day-old plants.
Figure 7Percentage of cells that are positive for pro-Collagen I in wild-type (WT), Fam134 knockout MEFs, and Fam134 knockout MEFs reconstituted with the respective wild-type or ∆LIR mutant Fam134 protein. n=150 cells each condition. Representative Western Blot of Collagen I in wild-type MEFs and Fam134 knockout MEFs reconstituted for the respective wild-type (WT) or ∆LIR mutant Fam134 protein. Vinculin has been used as reference for ratio calculation. The statistical significance was estimated by unpaired Student's t test. All data are represented as mean ± s.e.m. of n=3 biological experiments and the statistical significance is defined as * p<0.05, ** p<0.01, *** p<0.001. Representative of total cell lysate and the corresponding densitometric analysis of Collagen I in wild-type MEFs and Fam134 knockout MEFs reconstituted for the respective wild-type (WT) or ∆LIR mutant Fam134 protein. Vinculin has been used as reference for ratio calculation. The statistical significance was estimated by unpaired Student's t test. All data are represented as mean ± s.e.m. of n=3 biological experiments and the statistical significance is defined as * p<0.05, ** p<0.01, *** p<0.001. Representative Western Blot (D) and the relative bar plot (E) showing collagen expression in Fam134b knockout MEFs overexpressing wild type and the ∆LIR Fam134a protein. Representative Western Blot (F) and the relative bar plot (G) showing collagen expression in Fam134b knockout MEFs overexpressing wild type Fam134a and Fam134c. Representative Western Blot (H) and the relative bar plot (I) showing collagen expression in Fam134c knockout MEFs overexpressing wild type Fam134a and Fam134b. Representative Western Blot (J) and the relative bar plot (K) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134b. Representative Western Blot (L) and the relative bar plot (M) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134c.
Identification of novel category of FFAT motifs regulated by phosphorylation.Figure 1A: Schematic representation of STARD3, FIP200, MIGA2, PTPIP51, KCNB1 (Kv2.1) and KCNB2 (Kv2.2) proteins and sequence of their Phospho-FFAT motif (7 core residues and 4 upstream and downstream residues). Note that the 4th position of the FFAT (boxed) of these proteins is occupied by a serine or a threonine residue while conventional FFATs have an aspartic acid. Blue rectangles represent transmembrane helices. TPR: Tetratrico peptide repeat; LDT: lipid droplet targeting domain; BTB: Potassium channel tetramerisation-type BTB domain. B: Sequence of the peptides used for the pull-down assays. The peptides are composed of an amino-terminal biotin, a linker sequence and the FFAT sequence of STARD3 (residues 200-216), FIP200 (residues 725-741), MIGA2 (residues 286-302), PTPIP51 (residues 151-167), Kv2.1 (residues 584-600), Kv2.2 (residues 599-615), and ORP1 (residues 469-485) either without or with a phosphorylated residue (serine or threonine) at position 4 of the core FFAT motif. The negative control peptide is composed of a random sequence. C: Western blot analysis of proteins pulled down using the peptides input fraction corresponds to HeLa cell total protein extract. Bound proteins were analyzed using anti-VAP-A, anti VAP-B and anti-MOSPD2 antibodies. Actin was used as a loading control. D: Western blot analysis of proteins pulled down using peptides comprising WT and mutant (D478S and D478pS) ORP1 FFAT sequence peptides Bound proteins were analyzed using anti VAP-A, anti-VAP-B and anti MOSPD2 antibodies. Actin was used as a loading control. Data information: Acidic (D or phosphorylated residues (pS alcoholic (S residues are in red, green and blue, respectively; the other residues are in black. E: Venn diagram of VAPs and/or MOSPD2 partners with FFAT scores between 0 and 3. Proteins with a Phospho-FFAT and a conventional FFAT are shown in green and red, respectively. A total of 42 proteins possesses both a Phospho and a conventional FFAT. Consensus sequences of the 110 Phospho-FFAT (left) and 94 conventional FFAT motifs (right) identified are shown as sequence logos. Data information: Acidic E) or phosphorylated residues pT), alcoholic (S and T), and aromatic (F and Y) residues are in red, green and blue, respectively; the other residues are in black. F: List of selected VAP-A, VAP-B or MOSPD2 partners having a potential Phospho-FFAT motif.
dBruce colocalizes with the autophagic marker Atg8a-GFP in the nurse cells during late oogenesis and is accumulated in autophagy germline mutants.Figure 4.dBruce colocalizes with the autophagic marker Atg8a-GFP in the nurse cells during late oogenesis and is accumulated in autophagy germline mutants. Confocal micrographs of late stage egg chambers expressing Atg8a-GFP and stained for dBruce. (A-C) Stage 10 (A), early stage 12 (B), and late stage 12 (C). dBruce colocalizes with Atg8a-GFP in punctuate structures during stages 12 and 13 (insets in B and insets and arrows in C) and not during stage 10 (insets in A). (D-G) Confocal micrographs of stage 14 wild-type (D) and autophagy mutant egg chambers (E-G) stained for dBruce. dBruce is accumulated in atg1, atg13, and vps34 mutant nurse cells (arrows). Draq5/Hoechst staining (blue) was performed to visualize the nuclei. Nurse cells are outlined with a white line. OC, oocyte; PhC, phase contrast. Bars, 10 µm.
FU downregulates Smaug levels and repressive effect.Figure 4(A and C) Effect of FUEE-SBR on Smaug phosphorylation and levels. Extracts of Cl8 cells transfected with λN-SNAP-smaug (25 ng) and SNAP-GUS-5BoxB (300 ng) plasmids in absence or with different amounts of FUEE expressing plasmid (ranging from 15 to 60 μg, as indicated) were analyzed by electrophoresis before quantification as above. See a reprenstive gel in Fig EV5A. The % of phosphorylated SNAP-Smaug protein (determined as in Fig 3) (A) are represented as a function of the levels of FUEE-SBR. The mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom. The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value= 0.05 (*). Here CLIP-GUS was used as an internal control. (B) Effect of FUEE-SBR on Smaug activity. The relative levels of reporter expression obtained in the same experiment as in (A-C) are represented as a function of the % of phosphorylated Smaug. Color of data points correspond to ng of transfected FUEE-SBR in A-C. (A and C) Effect of FUEE-SBR on Smaug phosphorylation and levels. Extracts of Cl8 cells transfected with λN-SNAP-smaug (25 ng) and SNAP-GUS-5BoxB (300 ng) plasmids in absence or with different amounts of FUEE expressing plasmid (ranging from 15 to 60 μg, as indicated) were analyzed by electrophoresis before quantification as above. See a reprenstive gel in Fig EV5A. The relative levels of of λN-SNAP-Smaug protein (C) (determined as in Fig 2) are represented as a function of the levels of FUEE-SBR. The mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom. The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value= 0.05 (*). Here CLIP-GUS was used as an internal control. (D) Effect of FUEE-SBR on Smaug subcellular localization.Representative fluorescent images of Cl8 cells expressing GFP-SmaugWT (1), FUEE-SBR-mCherry (2), FUDANA-SBR-mCh (3) or expressing GFP-SmaugWT together with FUEE-SBR-mCherry (4-4'') or GFP-SmaugWT together with FUDANA-SBR-mCh (5-5''). The merge images in 4" and 5" show GFP-SmaugWT in green and FUEE-SBR-mCh or FUDANA-SBR-mCh in red. (6) In presence of FUEE-SBR, 274 of a total of 294 cotransfected cells show a diffuse localization of Smaug and FU and no foci. In presence of FUDANA-SBR, 100% of the cotransfected cells (n=241) show Smaug and FU distributed in bodies and colocalized. 250 ng of plasmid were used for each construct. Scale bar (shown in D1, identical for all panels): 10μ
ACE2 expression is upregulated in the lungs from SARS-CoV MA15 infected C57Bl/6 mouse.Figure 4. ACE2 expression is upregulated in the lungs from SARS-CoV MA15 infected C57Bl/6 mouse.Intranasal instillation of 102, 103 PFU of SARS-CoV MA15 in 50 µl of PBS or mock-infected with PBS alone, lungs were harvested 24 hrs later and total RNA was isolated and subjected to microarray analysis. ACE2 expression data was retrieved from Gene Expression Omnibus (GEO) microarray database (NCBI Accession: PRJNA149057; GEO: GSE33266).
Figure 5A, HCT116 cells were treated with either the vehicle alone (-), 1 µM MPA or 10 µM MPA for 24 hrs, before p21 immuno-labeling, propidium iodide staining and FACS analysis. Cell cycle profiles of the total cell population (upper panel, left), the total intensity of p21 and propidium iodide were determined in 20,000 cells and plotted in a scatter diagram (lower panel, left). The specific cell cycle profiles are shown of p21high (upper panel, right) and p21low (lower panel, right) cell populations. Mean ± SD of the percentage of cells in each cell cycle phase of at least two independent experiments is shown in the right panels. B, HCT116 p21-/- isogenic cell lines were transfected for 24 hrs with an empty plasmid (EV) or a plasmid encoding a p21wt cDNA (p21-OE), then cells were serum-deprived for 16 hrs before the re-addition of serum for additional 24 hrs in absence or presence of 10 µM MPA. Cells were subjected to p21 immunolabeling, propidium iodide staining and FACS analysis. The results are representative of two independent experiments.
Fig.Figure 1A. Images of P4 (top) and P21 (bottom) Mpdz+/+ and Mpdz-/- mice. Arrowheads point to the domed foreheads of the latter. B. Mean weights of P18-P21 Mpdz+/+ and Mpdz-/- mice (n=8÷12, mean ± SD). C. Coronal, axial, and sagittal (top to bottom) PET images of P18-P21 Mpdz+/+ and Mpdz-/- mice. The emission intensity is shown as a 7-point temperature scale from black (0) to white (7). D. Mean PET emission intensities in the indicated brain sections (n=4, mean ± SD).
Figure 3A Measurement of the ventilatory mechanic by barometric plethysmography: dot plots showing inspiratory (A1) and expiratory times (A2), the relaxation time (A3) and the respiratory frequency (A4) in WT (n=8), mdx (n=11) and mdx/CD38-/- (n=9) mice. Data information: Each dot of the graphs represents a mouse. one value/mouse After normality and variance comparison tests, significance was assessed using: A1,A3 ANOVA followed by Fisher's LSD test; A2, A4 : Kruskal-Wallis followed by Dunn's test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001. B Images showing muscle fiber typology revealed by immunostaining. Localization of the slow MyHC (Type I) fiber and fast MyHCs (Type IIa and IIb/X) fibers, along with laminin (red) on transverse cross-sections from diaphragm of WT, mdx and mdx/CD38-/- mice. Scale bars: 100 µm. Histogram showing the percentage of I, IIa and IIb/X fiber type distribution in diaphragm of WT (n=7), mdx (n=6) and mdx/CD38-/- (n=7) mice. Data information: one value/mouse After normality and variance comparison tests, significance was assessed using: ; B: Chi-square test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001. C Fiber size distribution in the diaphragm of WT (n=7), mdx (n=6) and mdx/CD38-/- (n=7) mice. Data information: one value/mouse After normality and variance comparison tests, significance was assessed using: C: Kolmogorov-Smirnov test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001. D Images revealing the collagen (blue) by Masson's trichrome staining in diaphragm of WT, mdx, and mdx/CD38-/- mice. Quantification of collagen staining area (% total area) in the diaphragm of WT, mdx and mdx/CD38-/- mice (n=4 per group). Scale bars: 200 µm. Data information: Each dot of the graphs represents a mouse. D in duplicate; After normality and variance comparison tests, significance was assessed using: : ANOVA followed by Fisher's LSD test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001. E Embryonic myosin expression: immunostaining showing its localization along with laminin (green) on transverse cross-sections from diaphragm of WT, mdx and mdx/CD38-/- mice. Scale bars: 50 µm. Histogram showing the relative proportion of embryonic myosin area (% total area) in diaphragm of WT (n=7), mdx (n=8) and mdx/CD38-/- (n=8) mice. Data information: Each dot of the graphs represents a mouse. E one value/mouse. After normality and variance comparison tests, significance was assessed using: E: Welch's ANOVA followed by Welch's t-tests. Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.
Inverse regulation of pro-inflammatory pathways by miRNA regulators HuR (ELAVL1) and PP2A (PPP2CA) in mammalian macrophage cells A-C Interaction routes between HuR (ELAVL1) and its target gene PP2A (PPP2CA).Figure 6Interaction routes between HuR (ELAVL1) and its target gene PP2A (PPP2CA). Alternate mechanism or regulatory steps that could be involved in establishing the regulatory relationship between HuR (ELAVL1) and its target genes have been investigated. A brief outline of possible alternate routes of regulation between HuR (ELAVL1) and PP2A (PPP2CA) via intermediates at the protein-protein interaction, cellular signalling, or by miRNA have been outlined in panel A. effect of HA-HuR expression on PP2A protein levels effect of HA-HuR expression on PP2A RNA levels were scored. Expression profiles of HuR, PP2A and miRNA let-7a are connected to IL-6 expression in RAW 264.7 cells. Data obtained from experiments described in previous figures are summed up to plot the changes in IL-6 mRNA levels against time of LPS treatment along with changes in PP2A, miRNA let-7a and HuR (D). Change in expression of IL-6 is connected to changes in let-7a concentration. The mathematical equation fitting the curve are shown here. Y represents concentration of IL-6 and X represents miRNA let-7a concentration. A1 is the initial IL-6 levels and A2 is the changed level. LogX0 is the mid-point of value 0.34, a concentration of let-7a where Il-6 expression is reduced to half variations of IL-6 expression with the altered level of PP2A (black) and HuR (red). For both cases, the data were fitted to an equation, which is shown above. The parameter m denotes the expression level of PP2A/HuR which corresponds to the maximum IL-6 expression; w denotes the width of the distributions for both cases. For PP2A and HuR, the values of m were found to be 0.6 and 0.9 respectively and A is a constant (F).
Figure 4(A) Vacuoles were isolated from BJ3505 cells expressing the indicated versions of Vam3 and/or Nyv1. 10 µg of the organelles were analyzed by SDS-PAGE and Western blotting using antibodies to Vti1, Vam3 and Nyv1. (B) Localization in vivo. The indicated strains were grown logarithmically in synthetic complete (SC) medium. After 16 h at 30°C, when the final OD of each culture was about 1, the cells were observed by confocal fluorescence and differential interference (DIC) microscopy. Scale bar: 5 µm.
Suppression of Rubicon expression enhanced the maturation of autophagosomes and inhibited HCV RNA replication.Fig 2(A) Huh7.5 cells were transfected with the negative control siRNA (NC) or the Rubicon (Rb) siRNA for 48 hours and then infected with 1 m.o.i. of HCV. Western-blot analysis of cell lysates at different time points after HCV infection. Actin served as the loading control. (B) Huh7.5 cells were transfected with the negative control siRNA (NC) or the Rubicon (Rb) siRNA for 48 hours and then infected with 1 m.o.i. of HCV. Real-time RT-PCR analysis of HCV RNA at 24 and 48 hours post-infection. *, p < 0.05. siNC, negative control siRNA; siRb, Rubicon siRNA. Huh7.5 cells were transfected with the negative control siRNA (NC) or the Rubicon (Rb) siRNA for 48 hours and then infected with 1 m.o.i. of HCV. (C) Fluorescence imaging of RFP and GFP puncta in cells transfected with the control siRNA (top two panels) or the Rubicon siRNA (bottom two panels). Cells were fixed at 24 and 48 hours after HCV infection for the analysis. Boxed areas in merged images are enlarged and shown to the right. (D) Percentages of RFP puncta that were also positive for GFP in Huh7.5 cells treated with either the control siRNA or the Rubicon siRNA. The results represent the average of >50 cells.
a) Immunoblot analysis of LAMP-2A in CD4+ T cells obtained from 4-month-old or 22-month old mice, then left resting or stimulated for 24 h with anti-CD3 plus anti-CD28.figf5(a) Immunoblot analysis of LAMP-2A in CD4+ T cells obtained from 4-month-old or 22-month old mice, then left resting or stimulated for 24 h with anti-CD3 plus anti-CD28. (b) Immunoblot analysis of RCAN1 and Itch in CD4+ T cells obtained from 4-month-old or 22-month old mice (n = 3 per group), then stimulated for 24 h as in a (left), and quantification of protein in 22-month old mice, presented relative to that of 4-month-old mice, set as 100%. *P = 0.044 (t-test). (c) Immunoblot analysis of LAMP-2A in total lysates of naive and memory CD4+ T cells left resting or activated for 24 h as in a. (d) Immunoblot analysis (bottom) of LAMP-2A in total lysates of naive and memory CD4+ T cells obtained from 4- or 22-month-old mice and left resting (R) or activated for 24 h as in a (A); above, quantification from results below (n = 3 mice per group). *P = 0.009 (t-test). (e) Electrophoretic mobility-shift assay of the binding of NFAT (N) and free probe (FP) in nuclear extracts of CD4+ T cells isolated from 3-, 12- or 22-month-old mice and activated for 4 h as in a. (f) ROS production in CD4+ T cells isolated from 4- or 22-month-old mice (n = 3 per group) and left resting or activated for 12 h as in a, assessed by flow cytometry and presented as the mean fluorescence intensity (MFI) of a fluorogenic probe for oxidative stress (CellROX Green). (g-i) Immunoblot analysis of LAMP-2A (g) and analysis of the proliferation (h) and IL-2 production (i) of TH1 cells obtained from 4- or 22-month-old mice and transduced with retrovirus expressing GFP alone (RV-GFP) or human LAMP-2A and GFP (RV-LAMP2A), sorted by GFP expression, cultured for 2 weeks and then stimulated for 24-48 h as in a (assessed as in Fig. 2c (h) or Fig. 2b (i)). *P 0.05 (analysis of variance with Tukey's post-test). (j) Immunoblot analysis of LAMP-2A in total lysates of CD4+ T cells obtained from two human donors (A and B) and left resting or activated for 24 or 48 h as in a (above lanes). (k) Real-time PCR analysis of LAMP2A mRNA in naive and memory CD4+ T cells obtained from young human donors (26 ± 2 years of age; n = 3) or old human donors (72 ± 6 years of age; n = 6) and activated for 24 h as in a; results were calculated by the change-in-cycle-threshold method (2−ΔCt) relative to those of the gene encoding β-actin. *P = 0.047 and **P = 0.0033 (Mann-Whitney).
Figure 2(A) Strategy for differential phosphoprotemics of HaCaT GALNT KO cell lines, where the Gaussian distributions and the Venn diagram represent selection criteria. (B) Analysis of differentially phosphorylated peptides. Upregulated or downregulated phosphopeptides in GALNT KOs were considered significant, if phosphopeptides were identified in both clones more than 2x SD away from the median, and given the same phosphopeptides were within the normal variation of two wild type biological replicates. The Venn diagram illustrates the selection criteria, where the color scheme corresponds to the Gaussian distribution illustration above. The bar graph depicts numbers of unique differential phosphosites in the individual GALNT KOs. (C) The Venn diagram represents overlap between differential phosphosites identified in the different GALNT isoform KOs. (D) GO terms (DAVID) associated with phosphoproteins identified in the 3 differential datasets. p value (Benjamini multiple comparison adjusted) of 0.05 is marked with a dashed line.