EMBO/sd-character-level-ner huggingface dataset:
Figure 1A. Two routes of MT polymerization in the presence of DCX are considered: direct nucleation, involving stabilization of early tubulin oligomers by DCX; stabilization, involving DCX binding only between straight PFs of a complete MT lattice. Question marks, unknown mode of DCX binding to tubulin/MT. B. Human DCX schematic, sequence alignment of the DC domains from human isoform 2 (Uniprot ID: O43602-2) with indication of secondary structures and 3D structure alignment, in which selected amino acid positions are indicated using single-letter codes. Sequence identity is 24.1 % by Clustal 2.1 (Larkin et al., 2007). H, α-helix; S, β-strand. C. List of DCX constructs with their abbreviated names and a summary of their activity. Construct boundaries are indicated with residue numbers. +, WT activity; +/-, impaired activity compared to WT; ++, enhanced activity compared to WT; -, no activity; nd, not determined. D. Plot of initial MT polymerization velocities at 37 °C in the presence of increasing concentrations of WT or NN. Tubulin concentration = 5 μM. Each point represents an average of 3 independent measurements of the rate of turbidity increase measured at 500 nm wavelength (OD500/min). Fitted are sigmoidal dose-response curves: Y = YB + ((YT - YB)/(1 + 10Xi - X)), where YB and YT are Y values (OD500/min) at the bottom and top plateaus, respectively, and Xi is the X value at the inflection point. Representative MT samples from 2:5 μM WT:tubulin or NN:tubulin incubations are shown in E and F, respectively. MT architecture distributions for WT and NN are shown with pie graphs. E, F. Representative low and high magnification images of MTs nucleated for 10 min at 37 °C from 5 μM tubulin, in the presence of: 2 μM wild-type DCX (WT) (E) or 2 μM NDC-NDC (NN) (F). G. Schematic of TIRF microscopy-based MT stabilization assay reported in H and I. Various concentrations of unlabelled DCX variants were added to 9 μM unlabelled tubulin mixed with 1 μM HiLyte Fluor™ 488-labeled tubulin in the presence of surface-immobilised HiLyte Fluor™ 488-labeled GMPCPP MT seeds; e-field, evanescent field. H. Representative kymographs with arrowheads indicating MT catastrophes. I. Quantification of MT catastrophe frequency in the presence of different DCX constructs observed with MT stabilization TIRF assay. Total free tubulin concentration was 10 μM in all experiments. The basal (tubulin alone) catastrophe frequency level at this tubulin concentration is indicated with a grey bar.
Autophagy Loss Disrupts Rab11-Mediated Recycling of EGFR.Figure 6A: Glial shNf-1/shTp53 control and sgAtg7 cells were serum starved 4 hr then stimulated with 20 ng/mL TexasRed-EGF (TxRed-EGF) for 30 min. Scale bar: 10 μm. B: Quantification of TxRed-EGF puncta per cell, relative to the number taken up by control cells (in A). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001. C: Endocytosis rate of wild type (WT) EGFR was assayed in control, sgAtg7, or sgAtg16l1 glial shNf-1/shTp53 or EGFRvIII-expressing cells by cell surface biotinylation and one application of 2 ng/mL EGF for 15 min. Monensin treatment was added as indicated. Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001. D: Plasma membrane recycling rate of WT EGFR in control, sgAtg7, or sgAtg16l1 glial shNf-1/shTp53 or EGFRvIII-expressing cells was assayed by cell surface biotinylation and successive applications of 2 ng/mL EGF treatments for 15 min Monensin treatment was added as indicated. Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001. E: Glial shNf-1/shTp53 control and sgAtg7 cells stably overexpressing mCherry-Rab11 were serum starved 4 hr then stimulated with 20 ng/mL EGF for 15 min then fixed and stained by immunofluorescence against EGFR. White arrows indicate colocalisation. Scale bar: 10 μm. F: Quantification of Pearson's colocalisation coefficient between mCherry-Rab11 and EGFR (in E). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001.
Figure 2Tau expression in the cortex (A, C, E) and hippocampus (B, D, F) of 4-10-month-old mice was determined by quantitative RT-PCR (A, B). (A, B) hTau mRNA levels in tissue homogenates were determined by quantitative RT-PCR. Tau expression in the cortex (A, C, E) and hippocampus (B, D, F) of 4-10-month-old mice was determined by western blot analysis (C-F).(C, D) Western blots from Figure 1A and B were enhanced to better reveal tau fragments of ~40 kDa. (E, F) Quantitation of western blot signals for tau fragments of ~40 kDa detected with Tau5 in tissue homogenates. n = 3-4 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed unpaired t test (A, B) or by Welch's one-way ANOVA with post-hoc two-tailed Welch's t test (E, F). P values were Holm-adjusted. Values are means ± SEM.
Figure 2Stable expression of LOC113230 in SW-1116 and HCT116 cells. LOC113230 was introduced into the cells via viral infections followed by puromycin selection and the resulting cells as mass pools were examined for LOC113230 level and subsequently used for cell migration assay. Overexpression of LOC113230 inhibited cell migration in SW-1116 and HCT116 cells. Cell migration was detected by transwell assays. Scale bar represents 100 μM. Depletion of LOC113230 in Caco-2 cells using specific shRNAs and stable cells were established with puromycin selection. The resulting cells as mass pools were used for cell migration assays. Scale bar represents 100 μM. Overexpression of LOC113230 in SW-1116 cells inhibited lung metastasis. Left: Exponentially growing SW-1116 - LOC113230-Luc and SW-1116-Vector-Luc cells were injected into the tail vein of 8-week-old nude mice and intensities of lung metastatic tumor cells at the 8th week were analyzed by in vivo photon flux imaging equipment. The red circles in images are to help to see the tumors. Right: Box plot (central band: median; box limits: first and third quartile; whiskers: minimum and maximum) of lung photon flux at the 8th weeks. 5 ng/ml TGF-β and DMSO were applied to stimulate SW-1116 -Vector or SW-1116 -LOC113230 cells for 24 and 48 hrs, respectively. Cell migration was detected by transwell assays. Scale bar represents 100 μM.
knockout results in the accumulation of poly-NEDD8 A (Left) Western blot analysis of large scale HALO-NEDP1 pulldowns from HEK 293 NEDP1 KO lysates.Figure 2(Left) Western blot analysis of large scale HALO-NEDP1 pulldowns from HEK 293 NEDP1 KO lysates. (Right) Diagram of mass spectrometry sample preparation. Table of the number of spectral counts from proteins with identified diGly- modified peptides, following pulldown by HALO-NEDP1 CA or the HALO-NEDP1 DAGC reduced binder control. NEDD8 is the protein most highly modified by NEDD8. Scatterplot of proteins identified by mass spectrometry analysis identifies NEDD8 and components of the NEDD8 conjugation pathway as being the most abundant proteins in HEK 293 NEDP1 KO lysates following NEDP1-CA pulldown. iBAQ analysis of proteins identified in A) are plotted as the log2 value of the enrichment ratio (mass spectrometry intensity of the HALO-NEDP1 CA pulldown over HALO-NEDP1 DAGC pulldown) versus the log10 value of the iBAQ intensity from the HALO-NEDP1 CA pulldown. Blue markers indicate known components of the NEDD8 pathway and red markers indicate proteins that have been identified as substrates of PARP-1 in the database of ADP-ribosylated proteins, ADPriboDB (Vivelo et al., 2017). Venn diagram of the proteins enriched by at least 6-fold following HALO-NEDP1 CA pulldown compared with proteins identified as PARP-1 substrates in the database of ADP-ribosylated proteins, ADPriboDB (Vivelo et al., 2017).
Figure 8(A) Immunoblots of protein lysates from Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (Tun.) (0.1 µg/ml) for the indicated times. Data are representative of three independent experiments.(B) Quantification of ATF4 protein levels in Null- or TFEB/3 knockout-MEF cells treated as indicated in (A) (mean ± SD of the ATF4 to actin protein ratio from three independent experiments, student's t-test versus the corresponding time point in Null-MEF cells, ns= not significant, **p < 0.01). (C) Cell viability was determined in Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (Tun.) (0.1 µg/ml) for the indicated times (mean ± SD of the percentage of viable cells compared to control from three independent experiments, one-way ANOVA analysis versus the corresponding time point in Null-MEF cells ***p < 0.001). (D) Chromatin condensation, an indication of cells undergoing apoptosis, was assessed in Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) for the indicated times. Confocal microscopy images were obtained from fixed cells stained with Hoechst 33342. Scale bar, 20 µm. Data are representative of three independent experiments. (E) Immunoblots of protein lysates from Null- or TFEB/3 knockout-MEF cells treated as described in (A). Data are representative of three independent experiments.(F-H) Quantification of CHOP (F), PUMA (G) and NOXA (H) protein levels in Null- or TFEB/3 knockout-MEF cells treated as indicated in (E) (mean ± SD of the indicated protein to actin ratio from three independent experiments, student's t-test analysis versus the corresponding time point in Null-MEF cells, ns= not significant, *p < 0.05, **p < 0.01). (I) adenovirus of protein lysates from ARPE-19 cells infected with either adenovirus expressing Null, TFEB-S211A or TFE3. Data are representative of three independent experiments.(J) Quantification of PUMA protein levels in ARPE-19 cells infected as indicated in (I) (mean ± SD of the fold change of the indicated protein to actin ratio from three independent experiments, one-way ANOVA analysis versus the adenovirus-Null infected cells, *p < 0.05, ***p < 0.001).
Autophagy is Required for the Maintenance of EGF-Mediated Signalling and Survival.Figure 7Glial shNf-1/shTp53 sgAtg7 (A) cells were either left untreated (+serum) or serum starved (-serum) 4 hr then stimulated 2 ng/mL EGF for 15 min before analyses by western blotting. Glial shNf-1/shTp53 sgAtg16l1 (B) cells were either left untreated (+serum) or serum starved (-serum) 4 hr then stimulated 2 ng/mL EGF for 15 min before analyses by western blotting. Glial shNf-1/shTp53 sgGal8 (C) cells were either left untreated (+serum) or serum starved (-serum) 4 hr then stimulated 2 ng/mL EGF for 15 min before analyses by western blotting. D: Glial shNf-1/shTp53 control and sgAtg7 cells were serum starved 4 hr then stimulated with 2 ng/mL EGF for the indicated times before analysis of EGFR signalling pathway activity by western blotting. E: Phosphorylated:total protein ratios for EGFR and AKT calculated from densitometry analyses of western blots were made relative to their respective points of maximal stimulation (100%) (for EGFR: t=5 min in control cells and for AKT: t=7 min in control cells) Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01. F: Glial shNf-1/shTp53 control and sgAtg7 cells were serum starved 4 hr with or without 20 ng/mL EGF 30 min stimulation before subcellular fractionation and analysis by western blotting. G: Radar chart indicating the relative levels of pAKT (pink line), pEGFR (light blue line), or total EGFR levels (dark blue line), in autophagy-deficient cell lines relative to their control counterparts. The following knockouts were introduced: glial cells (sgAtg7), HT29 (crATG7), MCF-7 (sgATG5), A549 (sgATG5), U2OS (sgATG5), and MEFs (Atg16l1-/-). H: Control or sgAtg7 glial shNf-1/shTp53 cells were serum starved for 24 hr in the presence or absence of 20 ng/mL EGF. Quantification of the percentage of propidium iodide (PI)-positive cells is shown. Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.
Figure 3A. Brain sections from 18-month-old ECrTgTau-Mapt0/0 and ECrTgTau mice were co-immuolabeled for human tau and misfolded tau (Alz50). Misfolded tau was only found in EC and DG neurons (white arrows) of ECrTgTau but not ECrTgTau-Mapt0/0 animals (n=4 sections/mouse, 3 mice/group). Scale bars 50 μm. B+C. Immunofluorescence labeling and stereological counting of microglia in entorhinal cortex (B) and astrocytes in hippocampus (C) indicated early signs of neurodegeneration in ECrTgTaumice. The significantly increased number of Iba1-positive microglia in the EC layer II/III of ECrTgtau mice (compared to WT) was partially rescued in ECrTgTau-Mapt0/0 mice (non-significant). The number of GFAP-positive astrocytes was similar across all genotypes (non-significant). Mean±SEM, n=4 sections per mouse, 3 mice/group, one-way ANOVA with Bonferroni correction. Scale bars 100 μm.
Figure 4A Protein was extracted from the left ventricle of wild-type or Cuedc2-/- adult mice (12 week-old) and subjected to immunoblot analysis (3 mice per group). B Wild-type or Cuedc2-/- mice were subjected to a heart ischemia for 30-minute in vivo, followed by reperfusion for the indicated times. Protein was extracted from the area at risk of left ventricle and subjected to immunoblot analysis. C GPX1 protein level was plotted using the immunohistochemical scores as described in methods. (left) representative images from immunohistochemistry staining for GPX1 in tissues from patients who suffered acute myocardial infarctions. (right) plot of GPX1 scores in each BZ and DZ (n = 9 patients). DZ, distant area; BZ, border zone. Scale bars, 250 μm (left). * p = 0.0039. D Primary neonatal mousecardiomyocytes stably expressing control or GPX1 shRNA by lentivirus infection were treated with H2O2 (1 mM) for 3 hours. Cell viability was detected by FACS. * p = 0.0001; ** p = 0.0275, n = 3 wells per group. NS, not significant. E Primary neonatal mousecardiomyocytes were transfected with lentivirus carrying control or GPX1 shRNA, and the efficiency of GPX1 knockdown was tested by immunoblotting. F Primary neonatal mousecardiomyocytes stably expressing control or GPX1 shRNA by lentivirus infection were subjected to H2O2 (1 mM) for 30 minutes and then stained with 5 μM CellROX® Deep Red Reagent and analyzed by flow cytometry. * p = 0.0056; ** p = 0.0218, n = 3 wells per group, n = 3 wells per group. NS, not significant.
Figure 1A. Verification of L-OHP resistance by CCK8 assay and schematic diagram of Ribo-seq in HCT116/L-OHP and HCT116 cells (n = 3, biological replicates, mean±SEM). B. Average lengths and numbers of identified lncORF, uORF, dORF and mORF by Ribo-seq. C. Scatter diagram of all kinds of sORF between groups by Ribo-seq. D. Expression distribution of lncORF in both groups by Ribo-seq. E. Bioinformatic analysis of ORF score and RRS for each ORF. F. Comprehensive analysis of differential expression and bioinformatics for identified lncORF. Shown in the heatmap, 56 potential translated lncORFs are related to drug resistance. G. Length distribution of encoded short peptides. H. Scatter diagram of lncRNA-seq in HCT116/L-OHP and HCT116 cells. I. Nine quadrant diagram of translatome and transcriptome correlation analysis. J. Venn diagram of the lncRNA different in Ribo-seq, high translation-potential by bioinformatics but indifferent in RNA-seq.
Figure 3Pom1 in vitro kinase assays with [ϒ-32P] ATP at five distinct Pom1 concentrations, ranging from 2.6 to 32ng/µl (1x to 12x). Loading of equivalent Pom1 amounts (40ng; see silver staining control) reveals higher incorporation of 32P upon higher Pom1 concentration, indicating an intermolecular reaction. Quantification is shown on the graph. Pom1as1-tdTomato in cells co-expressing Pom1-GFP (left) or Pom1KD-GFP (right) before and 1-2 min after addition of 3MB-PP1, as indicated. Before drug addition, Pom1 is strongly enriched at cell poles. After drug addition, Pom1as1-tdTomato is rapidly delocalized around the entire cell periphery (arrowheads) in cells co-expressing inactive Pom1KD-GFP, but not in cells co-expressing active Pom1-GFP, suggesting intermolecular phosphorylation (Hachet et al, 2011). Medial confocal planes are shown. Scale bar is 5µm. Quantification of data shown in panel B. The graphs on the left show the log-2 ratio of Pom1as1-tdTomato fluorescence levels at cell tip and cell middle relative to the medial Pom1-GFP (top) or Pom1KD-GFP (bottom) signal. Note that the of Pom1as1-tdTomato tip/middle log-2 ratios are lowered by 3MB-PP1 addition (red dots) relative to the non-treated samples (blue dots) in the cells co-expressing Pom1KD-GFP, but not in the cells co-expressing Pom1-GFP. A boxplot (right) of the residuals after correction of the effect of the GFP signal illustrates the significant effect of the drug treatment on Pom1as1-tdTomato localization in cells co-expressing Pom1KD-GFP (*, n=30+23, one-sided t-test, p-value =0.012) but not in cells expressing Pom1-GFP (n=30+30, two-sided t-test, p-value=0.39). + and - indicate presence or absence of 3MB-PP1.
Figure 8Ronin is dispensable for the development of the pluripotent lineage during the "quiet" pre-implantation embryogenesis, but it is critical for exit of embryo dormancy and for providing enough metabolic capacity for the post-implantation epiblast to support cell proliferation and morphogenesis.
Tgif1 inactivation promotes PDAC metastasis A Representative pictures of dissected abdominal region of KC and KTC mice.Figure 2Representative pictures of dissected abdominal region of KC and KTC mice. Yellow arrows indicate metastatic nodules in liver. Yellow dotted lines show the pancreas. FFPE sections of pancreas, liver, lungs or lymph-nodes from control, KC or KTC mice were stained with H&E or immunostained with anti-Vimentin antibody and revealed by IF. Representative pictures at 4x (Lymph node, H&E), 10x (Liver, H&E) and 20x (Pancreas, H&E; Lung, H&E; Lymph node, IF-Vimentin) are shown (n=18 to 30). Scale bars, 1,000 μM (Lymph node, H&E), 400 μM (Liver, H&E) and 200 μM (Pancreas, H&E; Lung, H&E; Lymph node, IF-Vimentin). Tumor volume and metastasis in live KCLuc and KTCLuc mice were analyzed by IVIS bioluminescence. Following imaging live animals, pancreas, brain, liver and lung were dissected and analyzed again by IVIS bioluminescence (n= 6, 5 out of 6 mice developed metastasis to liver and lung). Total cell lysates from pancreas of control, KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to pSmad2, Smad2 or JunB (n=6). Expression of JunB in pancreas from control, KC or KTC mice (same as in D) was analyzed by qRT-PCR (n=6). Data are expressed as mean ± SEM. **p< 0.01; based on a two-tailed Student's test.
Effect of YAP on the proliferation of normal and cancerous cervical cells.Figure 2A, C, E Western blot analysis showing levels of YAP and phosphorylated YAP in ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (A); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (C); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (E). β-Actin was used as a protein loading control. B, D, F Growth curves of YAP-overexpressed ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (B); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (D); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (F). Each point represents the mean ± SEM (n = 4). ***P < 0.0001, ME180-MXIV vs. ME180-YAP cells and ME180-MXIV vs. ME180-YAPS127A cells on day 8. ***P < 0.0001, HT3-MXIV vs. HT3-YAP cells and HT3-MXIV vs. HT3-YAPS127A cells on day 8. ***P < 0.0001, Ect1-MXIV vs. Ect1-Ect1-YAPS127A cells on day 4. *P < 0.0074, Ect1-MXIV vs. Ect1-YAP cells on day 4. G Western blot showing YAP levels in non-targeting control siRNA (siCtrl)- and YAP siRNA (siYAP)-transfected HT3 cells. H Proliferation of HT3 cells treated with control (siCtrl) or YAP siRNA (siYAP). Each point represents the mean ± SEM (n = 5). ***P < 0.001 compared with siCtrl (siCtrl vs. siYAP, P = 0.0002).
Reduced OPA1 levels in non‐fusing mitochondria and mitochondria inside APs.Figure 5(A) Relative immunofluorescence of OPA1 in INS1 cell mitochondria correlates to their fusion capacity. Cells (n=6) were photoactivated (∼10-15% of cell mitochondrial mass) and then returned to the incubator for 2 h before fixation. Based on their ability to share and thereby dilute the photoconverted mtPA‐GFP, mitochondria were classified as fused (n=54) in which GFP FI was lower than 2 times the average and non‐fused (n=10) in which GFP FI was at least 200% above the average (in the same manner as described for Figure 1). In each cell, OPA1 FI of non‐fused mitochondria was normalized to that of mitochondria that had diluted mtPA‐GFP through fusion. OPA1 FI is reduced in non‐fusing mitochondria (P=0.003). A set of FI‐coded images of mtPA‐GFP, mtDsRed and OPA1 immunostaining are shown. Scale bar, 2 μm. (B) OPA1 FI, but not mtDsRed FI, is reduced in mitochondria undergoing autophagy. INS1 cells expressing mtDsRed and LC3:GFP were treated with 0.2 μM bafilomycin (45 min) or a cocktail of pepstatin A and E64d (90 min) and fixed. Mitochondria inside APs were defined by colocalization of mtDsRed and LC3:GFP. OPA1 FI in APs was normalized to its FI in mitochondria that were located outside APs (organelles positive for mtDsRed with subthreshold GFP FI). OPA1 FI within APs was 50% (*P0.001) and 54% (**P=0.003) upon treatment with bafilomycin or a cocktail of pepstatin A and E64d, respectively. Mitochondria inside APs had similar (96%) mtDsRed FI to those located outside APs (P=0.47). Scale bar, 2 μm. (C) The effect of OPA1 overexpression on the number of AP‐containing mitochondria. Imaging of INS1 cells already expressing LC3:GFP and mtDsRed was performed 48-72 h after adenoviral transduction with either OPA1 overexpression or control (mitoPA‐GFP). The number of AP‐containing mitochondria in the OPA1 overexpression (OE, n=6) was normalized to the control (*P=0.001). Images show representative cells from each group. Red, mtDsRed; green, LC3:GFP. In the control cell, note the colocalization of APs with mitochondria. Scale bar, 10 μm.
Figure 1Distribution of S. indica apoplastic proteins identified by LC-MS/MS analysis from different symbiotic stages (5, 10 and 14 dpi) and in relation to proteins identified in CF of S. indica axenically grown in CM. In total, 102 S. indica proteins were identified in the APF of colonized barley roots. Of these, 33 proteins were present at all three time points. Twenty proteins were unique at 5 days post infection (dpi), 4 at 10 dpi and 21 at 14 dpi. Heat map showing absolute counts of unique peptides for S. indica apoplastic proteins present at all three time points and in culture filtrate. S1/S2/S3 = biologically independent sample 1, 2 and 3; d = deglycosylated.