EMBO/sd-character-level-ner huggingface dataset:
Figure 6A. LORE interacts with HopAO1 by MBP pull-down assays. The recombinant GST-HopAO1 and the MBP-LORE-CD were subjected to MBP pull-down analysis. MBP and GST proteins served as the negative controls. The interacting proteins were revealed by anti-GST immunoblotting. The experiment was repeated three times with similar results. B. LORE interacts with HopAO1 by Co-IP assays. 35S promoter-driven LORE-T7 and HopAO1-FLAG were transiently co-expressed in N. benthamiana by agroinfiltration. Plant leaves were immunoprecipated with anti-FLAG beads and immunoblotted by anti-FLAG. Coimmunoprecipitated proteins were immunoblotted by anti-T7. The experiment was repeated three times with similar results. C. HopAO1 dephosphorylates LORE Y600 in vitro. The recombinant GST-HopAO1/HopAO1CS and GST-LORE-CD were subjected to in vitro phosphatase assays. Phosphorylation level of Y600 was detected by anti-pY600 immunoblotting. The experiment was repeated three times with similar results. D. HopAO1 dephosphorylates LORE Y600 in N. benthamiana. LORE-FLAG was transiently co-expressed with GUS-T7, HopAO1-T7 or HopAO1CS-T7 in N. benthamiana leaves mediated by Agrobacteria. The protein was immunoprecipitated and probed by anti-pY600 antibody. The experiment was repeated at least three times with similar results.   E. HopAO1 suppresses 3-OH-C10:0-triggered ROS burst. HopAO1 was ectopically expressed in Col-0 driven by estradiol-inducible promoter. 100μM estradiol was sprayed on Est:HopAO1 plants, and the leaf disc was used for ROS burst assays 12 hrs later. Values are means ± SD (n=6 biological replicates). F. HopAO1 inhibits 3-OH-C10:0-induced disease resistance. The Est:HopAO1 plants were pretreated with 1 μM 3-OH-C10:0 (C10) and100 μM estradiol, and then the plants were inoculated with 5×104 cfu/ml Pst DC3000 24 hrs later. The bacterial titer was analyzed at 3 dpi. Values are means ± SD (n=6; ANOVA; p < 0.01 G. Working model: LORE is phosphorylated at tyrosine600 (Y600) at a basal level. In perception of 3-OH-C10:0, LORE Y600 phosphorylation is elevated, which then trans-phosphorylates PBL34 and likely its paralogs. Phosphorylated PBL34 dissociates from LORE, leading to immune activation. In this event, PBL34 kinase activity is required. However, the bacterial effector HopAO1 targets LORE Y600 and dephosphorylate the tyrosine-phosphorylated Y600, which subsequently suppresses immune response in Arabidopsis.
Wnt7a regulates actin dynamics in CAD cells overexpressing βCaMKII.Figure 5Confocal images of untransfected and GFP-βCaMKII WT transfected CAD cells (green), treated or not with Wnt7a. Top: Cells were stained with phalloidin (red) and DAPI (blue). Middle: Phalloidin staining (white) of the cells is shown. Bottom: Pseudocolor images of cells stained with phalloidin highlight the differences in F-actin levels among the treatments. Scale bars represent 5 μm. Anti-actin immunoblot and bar graph showing changes in the ratio between G-actin (in supernatant fraction, s) and F-actin (in the pellet fraction, p) of CAD cells untransfected or transfected with GFP-βCaMKII WT plasmid and treated or not with Wnt7a. Confocal images of CAD cells transfected with GFP-βCaMKII WT plasmid (green) and treated or not with Wnt7a. Cells were fixed and then stained with phalloidin (red) and DAPI (blue). The insets (right panels) show a magnified image of the area depicted in the expanded field images (left). 3D reconstruction shows co-localization of GFP/phalloidin signals. Scale bars represent 10 µm.
a) Representative images of rat liver (FAO) cells immunostained with TSC2, TSC1 or Rheb (green) and PMP70 (peroxisome marker) or LAMP1 (lysosome marker) (red) antibodies.figf1(a) Representative images of rat liver (FAO) cells immunostained with TSC2, TSC1 or Rheb (green) and PMP70 (peroxisome marker) or LAMP1 (lysosome marker) (red) antibodies. Scale bars, 10 μm. (b) Pearson's correlation coefficient for TSC1 or TSC2 co-localization with PMP70 or LAMP1 calculated using Imaris software. Quantification was performed on 8-12 cells from each of the 4 independent experiments, giving rise to a total of 40 cells. All error bars represent s.e.m., ***P0.001. (c) Representative images using HeLa cells transfected with Myc-TSC1 and Flag-TSC2 wild type (WT) and stained with anti-Flag (green) and anti-Myc (red) antibodies. Scale bar, 10 μm. (d) Representative images of HepG2 cells transfected with control or TSC2 siRNA immunostained for TSC2 (green) and PMP70 (red). Individual boundaries are shown to identify cells with TSC2 knockdown (white) versus cells that retain TSC2 (yellow). Scale bars, 10 μm. (e) Corresponding immunoblots for HepG2 cells transfected with control or TSC2 siRNA showing the extent of knockdown (average over population of cells). (f) HepG2 cells from d were analysed for Pearson's correlation coefficient of TSC2 co-localization with PMP70 using Imaris software. Quantification was performed on 8-12 cells from each of the 4 independent experiments, giving rise to a total of 40 cells. All error bars represent s.e.m., ***P0.001. Uncropped images of western blots are shown in Supplementary Fig. S6. Source data for the statistical analysis are shown in Supplementary Table S1.
Dietary Restriction Increases the Level of Autophagy.Figure 1LGG-1::GFP-positive puncta labeling autophagic membranes [22] were counted in wild-type or in food-limited animals.(A) Micrographs of eat-2(ad1116) L3 larvae expressing GFP-tagged lgg-1/LC3. Arrow indicates autophagic focus. Magnification is indicated.(B) Average number of LGG-1::GFP-containing puncta in eat-2(ad1116) mutants and N2 wild-type animals (WT), p < 0.0001.(C) Average number of LGG-1::GFP-containing puncta in N2 wild-type, food-restricted animals grown in liquid media (WT, food limited) and N2 wild-type animals grown in liquid with a higher concentration of bacteria (WT, fully fed), p < 0.0001; see Methods.Between three and ten seam cells were counted in each of 20-40 animals using high-power microscopy and averaged. n, total number of seam cells observed. Error bars: ±SEM. p-Values were calculated as unpaired, two-tailed t-test. Animals were raised at 20 °C. Please see Table S1 for quantification of all data.
Figure 3MARK4 siRNA-mediated silencing increases the tubulin cytoskeletal area (A). (A) Data shown are representative of experiments performed in triplicate. Bar graph represents the average ± SEM of n ≥ 50 per technical replicate with 3 technical replicates per conditions. MARK4 siRNA-mediated silencing reduces random (B) migration in MDA-MB-231 (MDA), A549 and H1299 cells.Cells were transfected either with siRNA targeting MARK4 (M4) or a non-targeting control (NC) for 48 h before time-lapse imaging was performed for 18 h (B). (B-top panel) Plots show overlays of representative trajectories described. (B-bottom panel) The migration distance was quantified and represented as the normalised mean ± SEM. (C-top panel) Representative field of view from the bottom side of the transwell membrane. (C-bottom panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n=5 fields per condition). (B-C) Data are mean of three experiments ± SEM. (A-C) P values were calculated by t-test between the siRNA-transfected and the respective control condition (***P<0.001). MARK4 siRNA-mediated silencing reduces directed (C) migration in MDA-MB-231 (MDA), A549 and H1299 cells.(C-top panel). Cells were transfected either with siRNA targeting MARK4 (M4) or a non-targeting control (NC) for 48 h before transwell assay for 9h (C). Representative field of view from the bottom side of the transwell membrane. (C-bottom panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n=5 fields per condition). (B-C) Data are mean of three experiments ± SEM. (A-C) P values were calculated by t-test between the siRNA-transfected and the respective control condition (***P<0.001).
IKKβ is required for pDC IFN-I production (A, B) Ratio of CD86 expression or IFN-I production in 5.Figure 6 (A, B) Ratio of CD86 expression or IFN-I production in 5.2+ vs 5.1+ pDC isolated from MCMV-infected (A MBMC of each indicated type  (A, B) Ratio of CD86 expression or IFN-I production in 5.2+ vs 5.1+ pDC isolated fro CpG-stimulated (B) MBMC of each indicated type  (C) Ratio of IL-12- or TNF-producing cells in 5.2+ vs 5.1+ pDC of indicated MCMV-infected MBMC mice
Figure 4A Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from L4+48 hour animals of the indicated genotype. ****P<0.0001, ANOVA, Tukey's multiple comparison test compared to wild type. N=20 animals per genotype. Error bars indicate SEM. B Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from either L4+24 hour animals (left half of graph) or L4+48 hour animals (right half of graph) of the indicated genotype. Animals were either continually grown on regular OP50 food (gray bars) or switched to PA14 food at the L4 stage (orange bars). ****P<0.0001, ***P<0.001, **P<0.01, ANOVA, Tukey's multiple comparison test compared to wild type. N=20 animals per genotype. Error bars indicate SEM. C Survival curves (percentage of live animals assayed each hour after PA14 exposure) for the indicated mutants at 20oC. Plog-rank<0.0001. Median survival time was 90 hours for wildtype (N=304), 72 hours for dop-1(vs100) (N=180), 78 hours for elt-3(gk121) (N=120), 78 hours for pqm-1(ok485) (N=116), 78 hours for nhr-28(gk568) (N=123), and 42 hours for skn-1(tm3411) (N=33). Eight wild-type animals and six dop-1 mutants were censored (the animals crawled up the side of the plate and dessicated). The media contained 50 µg/ml of 5-fluoro-2′-deoxyuridine (FUdR to prevent the growth of egg offspring during the experiment. D Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from either L4+48 hour animals (left half of graph) or L4+72 hour animals (right half of graph) of the indicated genotype. Animals were either continually grown on regular OP50 food (gray bars) or switched to NGM plates containing 1 mM paraquat at the L4 stage (orange bars). ****P<0.0001, ***P<0.001, ANOVA, Tukey's multiple comparison test compared to wild type. #P<0.001 ANOVA, Bonferoni multicomparison as indicated by the bracketed lines. N=20 animals per genotype. Error bars indicate SEM. No UbG76V-GFP fluorescence was detected in wild type at L4+72 hours on either OP50 or switched to NGM plates containing 1mM paraquat.
Figure 1(A to D) 10- to 11-week-old mice were treated with vehicle control or AT845 at the vector doses indicated and followed for 12 weeks (N = 7 or 8 per cohort). (A) Vector copy number, (B) GAA levels, (C) GAA activity, and (D) glycogen content in cardiac and skeletal muscles. (C to D) Statistical analysis: two-way ANOVA, Dunnett's test. Data are presented as box-and-whisker plots with Tukey whiskers that show minimum, median, and maximum. Asterisks (*) indicate significant differences compared with untreated Gaa-/- mice (black) or wild-type mice (red). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P <0.0001. See also Appendix Table S1.
USP26 participates in sex chromosomes pairing by regulating the stability of TEX11.Figure 6A USP26 interacts with TEX11. pRK-FLAG-Usp26 and pEGFP-Tex11 were co-transfected into HEK293T cells. 24h after transfection, cells were collected for immunoprecipitation (IP) with anti-FLAG antibody and analyzed with anti-FLAG and anti-GFP antibodies, respectively. B USP26 modulates the ubiquitination of TEX11. pRK-FLAG-Usp26, pEGFP-Tex11, pCMV-HA-Ub were transfected into HEK293T cells. At 24h after transfection, cells were collected for immunoprecipitation (IP) with anti-GFP antibody and analyzed with anti-HA and anti-GFP antibodies, respectively. C The TEX11 protein was greatly reduced in the Usp26-/Y testis. Immunoblotting of TEX11 was performed in 6-month-old Usp26+/Y and Usp26-/Y testes. Actin served as the loading control. The X and Y chromosomes were unpaired in Tex11-/Y spermatocytes. Immunofluorescence analysis of SYCP3 (green), TRF1 (red), ATR (white) (D); SYCP3 (green), MLH1 (red), ATR (white) (E) was performed in in Tex11+/Y and Tex11-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrowheads indicate the X chromosome. The X and Y chromosomes were unpaired in Tex11-/Y spermatocytes. Immunofluorescence analysis of ; Chr X-FISH (green), Chr Y-FISH (red) and SYCP3 (white) (F) was performed in in Tex11+/Y and Tex11-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrowheads indicate the X chromosome. G Quantification of unpaired X and Y chromosomes in Tex11+/Y and Tex11-/Y mice (n= 4 independent experiments). Red dots indicate Tex11+/Y mice and green dots indicate Tex11-/Y mice. H Laggard chromosomes were observed in the metaphase I of Tex11-/Y spermatocytes. The arrowhead indicates the laggard chromosome. I The proportion of metaphase I spermatocytes exhibiting laggard chromosomes in Tex11+/Y and Tex11-/Y mice spermatocytes (n= 3 independent experiments). Red dots indicate Tex11+/Y mice and green dots indicate Tex11-/Y mice. J Tex11-deficient mice produced XY aneuploid spermatozoa. FISH assay of Chr X (green) and Chr Y (red) was performed in Tex11+/Y and Tex11-/Y spermatozoa. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome.
Figure 7A, B Western blot analysis of heart and quadriceps lysates using anti-GAA detects both the precursor GAA (110 kDa) and the mature lysosomal GAA (70 to 76 kDa) forms in muscle of NHPs treated with (A) AT845 (n=3) or (B) Cyno-AT845 (n=3) at 2×1014 vg/kg. Each tissue was analyzed in triplicate, and each respective symbol indicates use of a vehicle control heart tissue (^) or a vehicle control quadriceps tissue (*). (See also Appendix Table S3)
Figure 3Time from NEBD to exit in indicated conditions with cells treated with nocodazole Red circles represent cells still arrested in mitosis when the filming stopped. Time from NEBD to exit in indicated conditions with cells treated with taxol. Red circles represent cells still arrested in mitosis when the filming stopped. Mass spectrometry analysis of BubR1 IPs from the indicated cell lines. Relative protein quantification values (MaxLFQ, log10) are plotted across conditions. Data from analysis of 3 technical repeats of BubR1 purifications with standard deviation indicated. Estimated protein levels of Bub1 in the indicated conditions relative to parental cell lines. In Bub1 CR we only detected one peptide from three purifications. Standard deviation indicated. Representative still images of unperturbed mitosis for parental cells, Bub1 C cells and Bub1 CR cells with time in minutes indicated. CFP-Histone 3 was used as the chromosome marker. Scale bar, 5 μm.
Figure 8A, B, Flow cytometric analysis showing enhancement of material transfer, of protein (biological replicate n=12; technical replicate (n=3 per biological replicate) (A) and mitochondria (biological replicate n=3; technical replicate (n=3 per biological replicate) (B), in co-cultures treated with ROCK inhibitor Y27632. ME, material exchange. C, Confocal microscopy image of cryostat sections of C57BL/6J recipient retinas 21 days after transplant of C57BL/J6 photoreceptors infected with empty vector (n=5), RHOA overexpression (RHOA OE) (n=7) or RAC1 dominant negative (RAC1 DN) (n=7) lentiviruses. Quantification of MT in retinas transplanted with control, RHOA and RAC1 DN lentiviruses. D, GFP staining of whole-mounted retinas after transplantation with control, RHOA and RAC1 DN lentivirus infected donor cells (n=4 per group). Quantification shows a significant reduction in the average protrusion length per cell and in the total protrusion length per cell after RHOA OE and RAC1 DN infection compared to the empty vector control.
Aberrant silencing of EMX2 and de-repression of HOX genes in glioma patients A.Figure 4Expression of EMX2 in normal brain and GBM patient samples. Data sourced from the Repository of Molecular Brain Neoplasia Data (REMBRANDT). Four asterisks indicate p-value < 0.0001 (two-tailed Mann Whitney U test). Bars represent median ± interquartile range. Values are expressed relative to the mean of normal brain samples. N: 21 for normal brain, 214 for GBM. Note that the extent of EMX2 repression in GBM patients is likely underestimated due to the possible presence of normal adjacent tissue in the analysed samples, and due to the intrinsic background noise of microarrays, which limits detection of truly silenced genes. Relative expression of HOX genes in normal brain and GBM patient samples as detected by microarray analysis. Data sourced from REMBRANDT. Values are expressed relative to the median of normal brain samples. In the boxplots, the top, middle and bottom box delimiters represent the 75th, 50th and 25th percentiles of the data, respectively. Top and bottom whiskers show the 75th percentile + 1.5*interquartile range and 25th percentile - 1.5*interquartile range, respectively. One asterisk indicates p-value <0.05 (two-tailed Mann Whitney U test corrected for multiple comparison using Holm's method). N: 21 for normal brain, 214 for GBM. Data was not available for HOXD8. Covariance between EMX2 and EZH2 expression levels in GBM patient samples, as detected by microarray analysis. Data sourced from REMBRANDT. P-value and correlation coefficient (r) of the covariance are shown (Spearman rank correlation). Every dot is a patient. N: 214. Expression levels of EMX2 (top) and EZH2 (bottom) in tumour or adjacent normal regions laser microdissected from human GBM tumours as detected by RNA-seq. Data sourced from the Ivy Glioblastoma Atlas. FPKM: fragments per kilobase of transcript per million. The significance of the differential expression in normal and tumour regions across patients is indicated (two-way ANOVA). Bars represent mean ± SEM. N: 3 regions sampled for each GBM tumour, 2, 3, 2, 3, 2, 3, 3 and 1 regions for normal tissue of patients 1-8, respectively. Scoring of EMX2 and EZH2 mRNA staining intensity in human GBM tumour samples. Scoring system: - 0-0.5 foci/nuclei, + 0.51-0.75 foci/nuclei, ++ 0.76-1 foci/nuclei and +++ >1 foci/nuclei Hematoxylin and eosin (H&E) staining of a human GBM tumour. The black square represents the approximate location of the field shown in G (left image). Serial sections were used for H&E and RNA-FISH. Scale bar: 300 µm. Visualisation of EZH2 (green) and EMX2 mRNA (red) in a human GBM tumour by RNA FISH. Nuclei were counterstained with DAPI. Scale bar: 20 µm. Expression of EMX2 (left) and EZH2 (right) in glioma patient samples, grouped according to tumour grade, as detected by RNA-seq. Data sourced from the Chinese Glioma Genome Atlas. Four asterisks indicate p-value < 0.0001 (Kruskal-Wallis test). Bars represent median ± interquartile range. N: 109 for grade II -, 72 for grade III, 144 for grade IV. RPKM: Reads per kilobase of transcript per million.
PAI-1 counteracts the negative feedback.Figure 5A The complex between uPA and PAI-1 is an agonist of the uPAR-mediated cell adhesion to VN. RTCA analysis of 293/uPAR cells seeded on VN and treated with 10 nM sc-uPA, PAI-1 and uPA•PAI-1 or a combination of 10 nM tc-uPA and 10 nM PAI-1. The time of the addition is indicated by a vertical dashed line. B Morphology of 293/uPAR cells treated with different agonists and antagonists of the uPAR-VN interaction. 293/uPAR cells were seeded on VN-coated cover-slips, allowed to adhere and treated with 10 nM PAI-1, sc-uPA and uPA•PAI-1 or vehicle for 1 h. The cells were fixed and DIC light microscopy images recorded. Representative DIC images are shown. Scale bar, 10 µM. C Effect of the different treatments on 2D cell migration on VN. 293/uPAR cells were seeded in VN-coated wells and treated as in panel B. Cells were imaged by time-lapse microscopy. Cell migration was quantified using ImageJ. The curves represent the average migration speed of 50 or more individual cells for each condition. The time at which cells were treated is depicted with a vertical dashed line. D In the presence of PAI-1, plasminogen activation stimulates cell adhesion on VN. 293/uPARWT cells were seeded on VN-coated plates. Cells were treated with 10 nM sc-uPA and 10 nM PAI-1 either alone or in combination followed by the addition of 30 nM Plg and 100 nM a2AP or vehicle as control. A representative RTCA experiment is shown.
Defective proteostasis and accelerated OPA1 processing in brain and spinal cord of NYKO mice.Figure 7A TEM analysis of sagittal spinal cord sections of 6-7 week old WT and NYKO mice. Scale bars 500 nm. B Frequency distribution of mitochondrial lengths in spinal cords of 6-7 week old WT (182 mitochondria) and NYKO mice (192 mitochondria). Kruskal-Wallis test, *P < 0.05 C TEM analysis of cerebellar sections of 28-32 week old WT and NYKO mice. Scale bars 1 µm. D Frequency distribution of mitochondrial aspect ratios were calculated from WT (n = 210) and NYKO (n = 230) mitochondria. Mann-Whitney test, ****P < 0.0001. Data are presented as median values. Immunoblot analysis of forebrain, cerebellar and spinal cord lysates from 31-32 week old WT and NYKO mice (n = 3) using the indicated antibodies. SDHA and LONP1 were used to control for gel loading. * indicates unspecific antibody binding.
SPPL2c mediated cleavage of Syntaxin 5 impairs GnT-V trafficking in HEK293 cells.Figure 4A) Additional candidate SPPL2c substrates associated with vesicular trafficking were identified using the function "More" of the STRING 10.5 software. The type IV membrane proteins identified as candidate substrates in the proteomic analysis served as basis for the analysis. B) Processing of endogenous Syntaxin 5 (Stx5) and Syntaxin 6 (Stx6) by SPPL2c wt by Western Blot. Non-transfected cells (-), stably transfected but non-induced SPPL2c wt cells (n/i) and induced cells stably expressing SPPL2c D/A served as controls. Results are derived from one experiment, thus expression (SPPL2c) and loading (Calnexin) control are the same. Note, that Syntaxin 5 is cleaved by SPPL2c while Syntaxin 6 is not. C) Syntaxin 5 protein levels were down regulated and processing and maturation of ectopically expressed GnT-V, as well maturation of the endogenous glycoproteins Nicastrin (NCT) and SPPL2a was analyzed in Western Blot. Calnexin served as loading control and ectopic expression of SPPL2c WT was confirmed using a SPPL2c specific antibody. While GnT-V and Nicastrin maturation were similarly affected by Syntaxin 5 knock down and SPPL2c overexpression, SPPL2a maturation was hardly impaired in Syntaxin 5 knock down cells. D) Syntaxin 5 localization is impaired by overexpression of SPPL2c. Endogenous Syntaxin 5 was visualized in control cells without SPPL2c expression (Ctr) and in cells overexpressing SPPL2c wt or SPPL2c D/A using immunofluorescence. BiP was used as a marker protein of the ER and TGN46 as Golgi marker. White arrow indicating cells with disturbed Golgi and Syntaxin 5 staining. Scale bar 5 µm. E) Syntaxin 5 knock down induces mislocalization of GnT-V similar to SPPL2c overexpression Cells ectopically expressing GnT-V were treated either with non-specific siRNA (siCtr) or with siRNA targeting Syntaxin 5 (siStx5). GnT-V was visualized in immunofluorescence using anti-V5 antibody targeting its C-terminal tag and either BIP was co-stained as ER marker protein or TGN46 as Golgi marker protein. Scale bar 5 µm.