EMBO/sd-character-level-ner huggingface dataset:
Figure 6(A). Flowchart for the workflow to predict the candidate substrates of PTP-MEG2 during fusion pore initiation and expansion. A total of 51 proteins were enriched with the functional protein association network STRING and the text mining tool PubTator by searching the keywords "fusion pore", "secretory vesicle" and "tyrosine phosphorylation". These proteins were filtered with UniProt by selecting proteins located only in the membrane or vesicle, which resulted in 28 candidates. The Human Protein Atlas database was then applied to exclude proteins with no expression in the adrenal gland. Finally, we used the post-translational-motif database PhosphoSitePlus to screen candidate proteins with potential phospho-sites that matched our sequence motif prediction at the pY+1 or pY+2 positions. (B). After the bioinformatics analysis, a total of 12 candidate PTP-MEG2 substrates that may participate in fusion pore initiation and expansion and their potential phospho-sites were displayed. (C). The GST pull-down assay suggested that PACSIN1, MUNC18-1, VAMP7, SNAP25, DYNAMIN1 and DYNAMIN2 directly interact with PTP-MEG2. PC12 cells were transfected with plasmids of candidate substrates, including SYN1, MUNC18-3, PACSIN1, SCAMP1, MUNC18-1, PPP3CA, STX17, VAMP7, SYT7, SYT11, SNAP25, DYNAMIN1, and DYNAMIN2 stimulated with 100 nM AngII. The tyrosine phosphorylation of these proteins was verified by specific anti-pY antibodies (Appendix Figure S8A-E). The potential substrates of PTP-MEG2 in cell lysates were pulled down with a GST-PTP-MEG2-D470A trapping mutant and then detected by Western blot. White arrow stands for Western blot band consistent with the predicted molecular weight of the potential PTP-MEG2 substrate. (D-F). Co-immunostaining assays of PTP-MEG2 with potential substrates in the adrenal medulla. MUNC18-1, VAMP7 and DYNAMIN2 all showed strong co-localization with PTP-MEG2 after 100 nM AngII stimulation in the adrenal medulla. White arrow stands for co-localization of PTP-MEG2 with MUNC18-1, VAMP7 or DYNAMIN2. The Pearson's correlation coefficients for (D, E and F) were 0.61, 0.65 and 0.79 respectively. The co-immunostaining results of PTP-MEG2 with other potential substrates are shown in Appendix Figure S7.
Figure 1A, B. Depletion of Ccdc108 markedly affects bead movement by decreasing velocity Fluorescent beads were applied to the dorsal surface of Xenopus embryos. Bead tracks every 500 ms over 2 s are shown (A). Plot of average bead velocity shows significantly reduced bead flow in ccdc108 morphants, which can be fully rescued by co-injecting with Xenopus ccdc108 mRNA (B). 150 beads from 15 embryos for each condition. Arrowheads indicate beads that showed no movement on the embryo surface through the recording. Data information: Quantification data were collected from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are presented. C, D. Defective cilia formation in ccdc108 morphants. Embryos at stage 27 were fixed and stained with the acetylated tubulin (Ac-tub) antibody and/or Cep164 antibody. mRNA of a membrane-bound form of GFP (mGFP) was co-injected with each morpholino to indicate targeted cells. Representative confocal images (C) and 3D-SIM images (D) of the epidermis for each condition and plots show significantly reduced cilia number in Ccdc108 depleted embryos. (C) 20 images of 20 embryos and (D) > 80 MCCs from 6 embryos for each condition. Data information: Quantification data were collected from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are presented. E. Scanning electron microscope images of Xenopus embryos display defective cilia formation in Ccdc108 depleted MCCs. White arrowheads mark MCCs with reduced ciliation. Boxed areas are magnified to show the details. F. Defective cilia formation occurs in ccdc108 CRISPR mutants. Embryos at one cell stage were injected with Cas9 protein with or without the sgRNA against ccdc108 (108sg) and fixed at stage 27. Representative 3D-SIM images and plot show significantly reduced cilia number in ccdc108 CRISPR mutants. > 70 MCCs from 6 embryos for each condition. Cell membranes (mGFP, purple), cilia (Ac-tub, green) and basal bodies (Cep164, yellow) were labeled with indicated antibodies. Data information: Quantification data were collected from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are presented. G. In situ hybridization chain reaction (HCR) reveals that ccdc108 mRNA is specially detected in MCCs. Embryos at stage 35 were fixed and subjected to in situ HCR (red). Embryos were then incubated with the Ac-tub antibody (cyan) and DAPI (gray).
a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag–Parkin wild-type or pathogenic mutants.figf4a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag-Parkin wild-type or pathogenic mutants. Cells were treated with CCCP before immunostaining of Parkin with anti-Flag (green) and endogenous poly-ubiquitin with a specific antibody FK1 (red). (a) In contrast to pathogenic mutations, wild-type Parkin shows colocalization with poly-ubiquitin after 6 h of CCCP treatment at the site of condensed mitochondria. (b) After 24 h of CCCP treatment, K161N and R275W Parkin mutants colocalize with both poly-ubiquitin and mitochondria. In contrast, translocation-deficient Parkin mutants K211N, T240R and G430D lack the mitochondria-associated poly-ubiquitin signal. Representative images are shown. Scale bars, 10 μm. Complementary Parkin of untreated cells and quantification of colocalization of Parkin, mitochondria and poly-ubiquitin upon CCCP treatment are shown in Supplementary Information, Fig. S3a, b and c, respectively. (c, d) Parkin-directed mitophagy specifically involves linkage of ubiquitin through lysines 27 and 63. HeLa cells were transfected with EGFP-Parkin wild-type and ubiquitin lysine variants and treated with CCCP before immunostaining with anti-HtrA2/Omi, as a mitochondrial marker (pseudo coloured; blue) and anti-His (red) to detect ectopic ubiquitin. Parkin was visualized by its fusion to EGFP (green). (c) Note that only wild-type, K27 and K63 6×His-tagged ubiquitin variants are colocalized with condensed mitochondria and EGFP-Parkin after 6 h of CCCP treatment. All other ubiquitin mutants interfere with Parkin translocation to mitochondria. (d) Only co-expression of K27 or K63 ubiquitin recapitulates mitochondrial clearance similarly to 6×His-tagged wild-type ubiquitin after 24 h of CCCP treatment. Representative images are shown. Scale bars, 10 μm. Immunostaining of untreated cells and all other ubiquitin variant combinations tested are shown in Supplementary Information, Fig. S4a and b, c, respectively. WT, wild-type; PolyUb, poly-ubiquitin.
a,b) Survival of wild-type (Oregon R), yw, PGRP-LE112, PGRP-LC7454 and PGRP-LE112,PGRP-LC7454 flies after injection of wild-type L.figf1(a,b) Survival of wild-type (Oregon R), yw, PGRP-LE112, PGRP-LC7454 and PGRP-LE112,PGRP-LC7454 flies after injection of wild-type L. monocytogenes (a) or Δhly L. monocytogenes (b) at 28 °C. (c) Survival of wild-type, yw and PGRP-LE112 flies, as well as flies treated with RNAi targeting PGRP-LE with the hml-Gal4 driver (hml-Gal4>PGRP-LE RNAi) and PGRP-LE112 flies with hml-Gal4-driven PGRP-LE expression (PGRP-LE112,hml-Gal4>PGRP-LE), after injection of wild-type L. monocytogenes at 28 °C. NS, not significant; *, P 0.01 (Wilcoxon-Mann-Whitney test): P = 0.0006, 0.0043 or 0.0043, for yw versus PGRP-LE112, PGRP-LC7454 or PGRP-LE112,PGRP-LC7454, respectively (a); P = 0.0095, for yw versus RNAi targeting PGRP-LE (c). Data represent the average of four independent experiments with over 30 flies of each genotype examined at the same time.
mATG9/ATG16L1 Vesicle Fusion Is SNARE Dependent.Figure 4(A) HeLa cells were transfected with mStrawberry-ATG16 for 20 hr and treated for 10 min with N-ethylmaleimide (NEM; 100 μM) in full medium, fixed, and stained for mATG9. Histogram shows correlation between mATG9 and ATG16L1 (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001. (B) Schematic diagram of the in vitro fusion assay of ATG16L1 and mATG9 vesicles. Postnuclear supernatants (PNS) of cells expressing mStrawberry-ATG16L1 were mixed with PNS from cells expressing ATG9L1-GFP for 1 hr in the presence of ATP, ATP and NEM at 37°C, or with ATP on ice. The samples were fixed and mounted on a microscope slide with Mowiol 4-88 for confocal analysis. (C and D) (C) Representative fields of in vitro fusion assay of ATG16L1 and mATG9 vesicles. The quantification in (D) was performed on ten fields (similar to that shown in the figure) per experiment. The quantification is expressed as Pearson's coefficient. Error bar, SEM. ∗∗p < 0.001.
a) Neuronal expression of neither Hiw nor Rae1 rescues the synaptic terminal overgrowth phenotype caused by the loss of function of the other gene.figf7(a) Neuronal expression of neither Hiw nor Rae1 rescues the synaptic terminal overgrowth phenotype caused by the loss of function of the other gene. Quantification of the number of boutons in wild-type, Rae1EX28, Rae1EX28; Hiw rescue (Res) (Rae1EX28; elav-Gal4/UAS-GFP-hiw), hiwΔN and hiwΔN; Rae1 rescue (hiwΔN; elav-Gal4/UAS-NTAP-Rae1) larval NMJs (segment A3 muscle 6/7; n = 12, 12, 12, 7 and 11, respectively). (b) Representative confocal images of wild-type (BG380-Gal4/+; +/+; UAS-GFP-hiw/+) or Rae1 mutant (BG380-Gal4/+; Rae1EX28; UAS-GFP-hiw/+) ventral ganglions with UAS-GFP-hiw trangene expressed in neurons. The larvae were stained with antibodies to horseradish peroxidase (HRP, red) and GFP (green). Scale bar represents 10 μm. (c) Western blots of total proteins extracted from wild-type, hiwΔN, Rae1EX28 and Rae1EX28 rescue (Rae1EX28; elav-Gal4/UAS-NTAP-Rae1) larval brains probed with indicated antibodies. Full-length blots are presented in Supplementary Figure 11. (d) Western blot analysis on total proteins extracted from hiwΔN, wild-type (C155-Gal4/+) and Rae1 expression (C155-Gal4/+; UAS-NTAP-Rae1/+) larval brains. Full-length blots are presented in Supplementary Figure 12. (e) Quantification of Hiw protein levels in the wild-type and the Rae1 expression larval brains (*P 0.001, based on seven independent experiments). Arrows indicate a 38-kDa endogenous Rae1 protein and the arrowheads indicate the 58-kDa transgenic NTAP-Rae1 protein. Error bars denote s.e.m.
Beclin localizes to the TGN.Figure 3Beclin localizes to the TGN. HeLa (A, D, E and F), BNL CL.2 (B) and H‐4‐II‐E cells (C) were grown on cover slips. Cells were permeabilized with digitonin before (C, D and E) or after (A, B and F) fixation with 3% paraformaldehyde. Cells were double‐labeled with anti‐Beclin (A-F, left panels) and either anti‐syntaxin 6 (A and B, middle panels), anti‐TGN38 (C, middle panel), anti‐EEA1 (D, middle panel), anti‐LBPA (E, middle panel) or anti‐TfR (F, middle panel) antibodies. In the merged images (right panels), yellow indicates co‐localization. Bar, 10 μm.
Fig.Figure 3A. Representative images of cells of A. tumefaciens C58 strains ΔtssB, ΔtssLΔtssB, and ΔtdeiΔtssB cells each expressing TssB-GFP from pTrc200. Upper panel: phase contrast images; Lower panel: green fluorescence images. Examples of GFP streaks were indicated by white arrowheads. Scale bar: 2 μm. B. Quantification of TssB-GFP foci in different genetic background. The number of the fluorescent foci were counted using the "analysis particle" function in Fiji with a manually set threshold. The number of cells were counted manually. For each strain, a total of three images were obtained from one independent experiment and each image contained more than 300 cells for quantification and statistical analysis. One-way ANOVA followed by Turkey HSD test was used for statistical analysis. Data are mean ± SEM and two groups with significant differences (P=0.000272) are indicated with different letters (a and b). Similar results are reproduced in two independent experiments. C. Western blots of the isolated sheaths, which were prepared via ultracentrifugation of cell lysates from A. tumefaciens C58 wild type, ΔtssL, and Δtdei. T: total proteins from the cell lysate. P: pellet samples containing TssBC sheaths after ultracentrifugation. S: supernatant after ultracentrifugation. Proteins were analyzed in western blots probed with indicated antibodies. Fha serves as a cytoplasmic control. Molecular weight markers (in kDa) are indicated on the left. Similar results were obtained from at least two independent experiments. Data are from one independent experiment and reproduced in at least two independent experiments. D. Pellet samples of wild type and Δtdei were visualized with transmission electron microscopy (TEM). Sheaths are indicated by red arrowheads and flagella, which are distinguishable on the basis of smoother, more solid, and thinner structures (~13 nm in width), are indicated by green arrowheads [33]. Data are from one independent experiment and reproduced in at least two independent experiments. Scale bar: 0.5 μm (upper panel), 100 nm (upper panel).
Gata6 progenitors contribute to SG morphogenesis.Figure 3(A) Bright-field images of whole-mount WT and Gata6 cKO epidermis, showing abnormal SG upon loss of Gata6. Quantification of hypotrophic SG in WT, heterozygous (Het) and Gata6 cKO epidermal whole-mounts. Data are boxplots with indication of means. Box limits are minimum and maximum values. An average of 165 HF per mouse (from 3 to 4 mice per genotype) was analyzed. (*) p-value < 0.05; (**) p-value < 0.005; unpaired Student's t-test. Data information: Scale bars: 50 μm. (B) Schematic representation of plasmid constructs used for in utero lentiviral infection. Whole-mounts or sections of adult infected epidermis with empty vector (EV) or Gata6-ires-GFP (G6OE) lentivirus were stained for GFP and Fasn. In vivo overexpression of Gata6 leads to ectopic Fasn expression in the HF/SG unit. White dotted lines define SG, and yellow dotted lines define a cyst. Note that the cyst is mostly negative for Fasn in agreement with its SD-like phenotype. White arrows indicate GFP-positive infected cells. These cells are stained with Fasn only in G6OE condition. H&E-stained skin from G6OE mice shows a cyst with SG elements in the HF unit (black arrows). Staining for Gata6 (both endogenous and exogenous) shows that Gata6 expression occurs in a limited number of cells (representative image in upper right panel). Bottom left graph shows quantification of the percentage of clones labelled for both Fasn and GFP in the SG, HF and IFE compartments. Data are means ± SD and originate from 4 EV mice and 8 G6OE mice (average of 11 clones per mouse). (C) Whole-mounts adult infected epidermis with EV or G6OE lentivirus stained with LipidTOX. Ectopic Gata6 cells are not stained with LipidTOX indicating an incomplete sebaceous maturation. Data information: Scale bars: 50 μm. (D) Lineage tracing experiments in Gata6EGFPCreERT2:Rosa26-fl/STOP/fl-tdTomato (Gata6creER ROSA-dTom) mice. A single dose of 4OHT was injected in pregnant females at E16.5. Tail skin from pups was collected at P13. Representative example of whole-mount epidermis showing tdTomato labelled cells counterstained with Dapi (top left panel). Right panels show the different Z-stacks related to this whole-mount. Gata6 progenies are mainly found in the upper SG/JZ. Localization of Gata6 progenies (dTomato+) was quantified in 20-26 pilosebaceous units per mouse (N=4 mice) (bottom left panel). Data are means ± SD. As a control, quantification was also performed in Rosa26-fl/STOP/fl-tdTomato (ROSA-dTom) mice that were treated similarly to Gata6creER ROSA-dTomato mice. Data information: Scale bar: 25 μm.
Figure 1A. SF-1, FATE1 and β-tubulin protein levels shown in basal condition and after Dox treatment in H295R/TR, H295R/TRSF-1, H295R/TRFATE1 and H295R/TR N-FlagFATE1cells. B. FATE1 mRNA expression is increased in H295R/TRSF-1 cells by Dox treatment. The efficiency of FATE1 knockdown by specific (siFATE1) vs. control (siC) siRNA nucleofection is shown (mean ± SEM; n=4 with 3 replicates/experiment). C. Immunofluorescence microscopy showing endogenous FATE1 protein (red) induction of expression by Dox-treatment of H295R/TRSF-1 cells. SF-1 (green), DAPI (blue). Scale bars, 10 μm. D. Subcellular localization of endogenous FATE1 (red) and transfected fluorescent markers for Golgi, ER and mitochondria, respectively (green) in Dox-treated H295R/TRSF-1 cells. DAPI (blue). Scale bars, 10 μm. E. Subcellular localization of the endogenous FATE1 protein (green) in Dox-treated H295R/TRSF-1 cells costained with an antibody against the mitochondrial marker HSP60 (red). SF-1 (blue). Scale bar, 10 μm. F. Immunogold electron microscopy showing association of FATE1 with the mitochondrial outer surface in Dox-treated H295R/TR N-FlagFATE1 cells. Scale bar, 500 nm. G. Dox-treated H295R/TR N-FlagFATE1 cells were fractioned into nuclear (N), heavy membranes (HM), light membranes (LM) and cytosolic (C) fractions and localization of SF-1, FATE1, ribosomal protein RPL7, VDAC1 and GAPDH was revealed by immunoblot. H. Effect of increasing concentrations (0, 1, 10 and 100 μg/ml) of proteinase K and 0.1% Triton X-100 (TX-100) treatment of the mitochondrial fraction from Dox-treated H295R/TR N-FlagFATE1 cells on VDAC1, FATE1, TOM20 and cytochrome c. I. FATE1 and VDAC1 are associated with the pellet (membrane; P) fraction after high-speed centrifugation of the alkaline-extracted mitochondrial fraction of Dox-treated H295R/TR SF-1 cells, while cytochrome c is found in the supernatant (S).
Figure 1(A) Venn diagram of STK38 partners identified in both starvation and suspension conditions by proximity biotinylation assay coupled to mass spectrometry identification (B) Heatmap representation of common STK38 interactors identified in both starvation and suspension conditions according to their dynamic of association with STK38 (with a minimal fold increase of 1.38 ± 0.06). Unsupervised hierarchical clustering was generated based on their association fold using Pearson correlation. (C) Representation of the 10 most cellular component enriched terms of the two clusters (assessed by using the Gene Ontology (GO) database
, Mathematical model structure of IFNα-induced JAK/STAT signal transduction pathway The model structure is represented by a process diagram displayed according to Systems Biology Graphical Notation (Le Novere et al, 2009).Figure 2The model structure is represented by a process diagram displayed according to Systems Biology Graphical Notation (Le Novere et al, 2009). Negative regulators are depicted in red. c: cytoplasm, n: nucleus, dim: dimer, rec: receptor, a: active, OccGASbs: occupied binding sites containing gamma-activated sequence, OccGASbs+OccISREbs: occupied binding sites containing gamma-activated sequence and interferon-stimulated response element.
FoxO3 controls mitochondrial network.Figure 2(A) Imaging of FoxO3‐mediated modification of the mitochondrial network in soleus muscle of living mice. Adult soleus muscles were transfected with pDsRed2‐Mito and either c.a.FoxO3 or mock vector (control). Two weeks later, muscles were exposed and observed in situ using two‐photon microscopy. At least five animals per condition were studied. (B) Overexpression of c.a.FoxO3 induces mitochondrial changes and removal through autophagy. Muscle fibres were transfected by electroporation with YFP‐LC3, pDsRed2‐Mito and either c.a.FoxO3 or mock vector. Two weeks later, fluorescent fibres were analysed for mitochondrial distribution by confocal microscopy. A higher magnification of the square is depicted on the right panels. White arrows point autophagosomes engulfing the mitochondria. (C) Overexpression of c.a.FoxO3 induces reduction in myofibre size and decrease in SDH staining. Left image: immunostaining for FoxO3 shows atrophic fibres, which over‐expressed FoxO3 (red) and are labelled by asterisks. Nuclei are stained by Hoechst (blue). Right image: histochemistry for succinate dehydrogenase (SDH) on serial section. Note that fibres positive for FoxO3 are negative for SDH staining (asterisks). (D) Electron micrograph of a control and c.a.FoxO3‐expressing fibres. Muscles were processed with standard fixation‐embedding procedure. Black and white arrows indicate subsarcolemmal and intermyofibrillar mitochondria, respectively. Note that subsarcolemmal mitochondria are reduced in FoxO3 overexpressing fibres, whereas intermyofibrillar mitochondria are smaller and paler than control ones. Red arrowheads show a vesicle containing mitochondria. The image is representative of the FoxO3‐transfected fibres.
Figure 3(A) Oxygen consumption of WT and isogenic mutant strains was measured with Clark oxygen electrode. Cells were grown in MHB-ca medium until exponential phase (OD600 = 0.6) or until late stationary phase (24 h). Cells were washed with PBS and respiration initiated by the addition of 20 mM glycerol. Data are expressed as average ± SD of four independent experiments. *** denotes p < 0.001 compared to WT via two-way ANOVA with Tukey's posttest. (B) NADH/NAD+ ratios were measured in cells grown aerobically in MHB-ca medium for 20 h were measured. 100% corresponds to a NADH/NAD+ ratio of 0.003. Data are expressed as average ± SD of four independent experiments. * Denotes p < 0.05, and *** denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest. (C) Acetate concentration was monitored in supernatants of WT and isogenic ndh strains at 7 and 24 h of growth in MHB-ca medium. Data are expressed as average ± SD of three independent experiments. ** Denotes p < 0.005 and *** denotes p < 0.001 compared to WT via two-way ANOVA with Tukey's posttest. (D) MKH2/MK ratios were measured in cells grown aerobically for 20 h in MHB-ca medium. Data are expressed as average ± SD of three independent experiments. * Denotes p < 0.05 compared to WT via one-way ANOVA with Dunnett's posttest.
is required for enzymatic activation (A) The domain organization of DDX43 and detail of the mutants.Figure 4(A) The domain organization of DDX43 and detail of the mutants. The conserved KH domain and DEAD-box helicase domain are indicated. D399 and E400 are highly conserved residues within the ATPase active site in the helicase domain. (B) Quantification of ATPase activity of DDX43 variants. The graph on the right indicates the ratio of the hydrolyzed ATP produced by DDX43 mutants to that of the WT DDX43. Data represent the mean+/- standard deviations. n=3 independent experiments. *p < 0.002 as determined with the t-test. (C) Immunoprecipitation of Ago3 with Flag-tagged DDX43 WT and its D399A and E400Q mutants. Ago3 and DDX43 were detected by western blotting using anti-Ago3 and anti-Flag-antibodies, respectively. Flag-EGFP was used as a negative control. The E400Q mutation in DDX43 impairs the interaction between Ago3 and DDX43. IgG h.c.: IgG heavy chain. (D) Immunoprecipitation of Ago3 with Flag-tagged DDX43 WT and its ∆KH mutant lacking KH domain. Ago3 and DDX43 were detected by western blotting using anti-Ago3 and anti-Flag-antibodies, respectively. Flag-EGFP was used as a negative control. The ∆KH mutant maintains affinity to Ago3. IgG h.c.: IgG heavy chain. (E) RNA-binding activity of DDX43 variants as analyzed by EMSA. Upper: The GIDN mutant was substituted G90 and I96 conserved within the KH domain to Asp and Asn, respectively. Lower: A 40-nt single-stranded RNA was 5′ end-labeled with 32P and incubated with DDX43 variants in the presence or absence of 0.1 mM ATP. Supershift means non-canonical complex. (F) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage. Unwinding activities of DDX43 WT and its D399A, E400Q, and ∆KH mutants are shown. RNAs cleaved by Ago3 are shown as "Cleaved RNA". (G) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage. Unwinding activities of DDX43 WT and its GIDN mutant are shown. RNA cleaved by Ago3 is shown as "3′ cleaved RNA".