EMBO/sd-character-level-ner huggingface dataset:
Phosphoinositide binding inhibits LAPTM4B interaction with Hrs and compromise the inhibitory effects of LAPTM4B in EGF‐stimulated EGFR degradationControl or LAPTM4B knockdown MDA‐MB‐231 cells were starved and stimulated with 100 ng/ml EGF for 30 min, and whole‐cell lysates were subject to co‐immunoprecipitation (co‐IP) assay.Figure 6A Control or LAPTM4B knockdown MDA‐MB‐231 cells were starved and stimulated with 100 ng/ml EGF for 30 min, and whole‐cell lysates were subject to co‐immunoprecipitation (co‐IP) assay. B Control or LAPTM4B knockdown MDA‐MB‐231 cells were transfected with His‐tagged ubiquitin, starved, and stimulated with 100 ng/ml EGF for 30 min. Total ubiquitinated proteins were purified from whole‐cell lysates by Ni‐NTA agarose and analyzed by Western blot. C Control or LAPTM4B‐WT/6RQ‐overexpressing MDA‐MB‐231 cells were transfected with His‐tagged ubiquitin and myc‐tagged Hrs, starved, and stimulated with 100 ng/ml EGF for 30 min before whole‐cell lysate harvest. Total ubiquitinated proteins were purified by Ni‐NTA agarose and analyzed by Western blot.D Quantification of Hrs ubiquitination levels from the Western blot in (C) (mean + SD; n = 5). E The co‐IP of Myc‐Hrs with Flag‐LAPTM4B‐WT or 6RQ mutant in HEK293 cells. F The co‐IP between Hrs and Nedd4 with Flag‐LAPTM4B‐WT or 6RQ mutant overexpression in HEK293 cells. G The effects of LAPTM4B‐6RQ mutant overexpression on EGF‐stimulated EGFR degradation and signaling. Flag‐LAPTM4B‐WT or 6RQ mutant overexpression was accomplished by lentivirus‐mediated infection approach. Cells with low levels of expression were selected as polyclonal pools for comparison. Control or overexpressing cells were starved and stimulated with 100 ng/ml EGF for indicated time periods and whole‐cell lysates were analyzed by Western blot. H Quantification of EGFR and pAKT levels from the Western blot in (G) (mean ± SD; n = 5). I The PY motif mutant LAPTM4B‐2PA loses interaction with Nedd4 but keeps interaction with Hrs in HEK293 cells cotransfected with the indicated proteins. J The effects of LAPTM4B‐2PA mutant overexpression on EGF‐stimulated EGFR degradation and signaling. Overexpression of Flag‐LAPTM4B‐WT or 2PA mutant overexpression was accomplished by lentivirus‐mediated infection approach. Cells with high expression of Flag‐LAPTM4B were selected as polyclonal pools for comparison. Cells were starved and stimulated with 100 ng/ml EGF for the indicated time periods, and whole‐cell lysates were analyzed by Western blot.K Quantification of EGFR degradation from Western blot in (J) (mean ± SD; n = 3).
Endogenous miRNA activity is detected in bovine and porcine oocytes.Figure 2(A) bta-miR-10b and ssc-miR-205 miRNAs efficiently repress microinjected NanoLuc luciferase reporters carrying a perfectly complementary (1x perfect) miRNA binding site or four partially complementary (bulged) miRNA binding sites. Error bars = SD. Each data point represents a value obtained from five microinjected oocytes. Data were collected in two microinjection sessions, each microinjection session used a new batch of in vitro transcribed reporter mRNAs. Data information: asterisks indicate statistical significance (p-value) of one-tailed t-test, (*p < 0.05, **p < 0.01, ***p < 0.001). (B) De-repression of predicted ssc-miR-205 targets. In three independent microinjection experiments, ssc-miR-205 was inhibited by a microinjected antisense oligonucleotide inhibitor and target levels were assessed 24 hours after microinjection by RT-qPCR. Error bars = SD. Data information: asterisks indicate statistical significance (p-value) of one-tailed t-test, (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Inhibition of ssc-miR-205 by a microinjected antisense oligonucleotide inhibitor results in reduced development to the blastocyst stage. Shown is development of microinjected oocytes to the blastocyst stage relative to water-injected oocytes. Five independent experiments (represented by individual data points) were performed, ~50 porcine oocytes were microinjected in each group in each experiment. Difference between let-7a and miR-205 inhibitors effects is statistically significant (two-tailed paired t-test p-value = 0.016). Error bars = SD. Data information: asterisks indicate statistical significance (p-value) of one-tailed t-test, (*p < 0.05, **p < 0.01, ***p < 0.001).
Direct chitin TLR2 binding and immune activation can be blocked by SSL3 or short chitin oligomers.Figure 4Flow cytometric quantificatio of Alexa647-labeled C10-15 interaction with mTLR2-Fc Protein (n=4 each) Data information: Quantification sub-panel represent data (mean+SE combined from 'n' biological replicate mic I one representative of 'n' independent experiments is show p<0.05 according to Mann-Whitney U (A microscale thermophoresis (MST) analysis (B) of Alexa647-labeled C10-15 interaction with mTLR2-Fc Protein (n=4 each) I one representative of 'n' independent experiments is show (C) Flow cytometr of Candida albicans stained with control IgG or mTLR2-Fc anti-Fc-Alexa594, together with ConA-Alexa488 and CFW (n=4 each). Scale bar = 5 μm Data information: Quantification sub-panel represent data (mean+SE combined from 'n' biological replicate mic I one representative of 'n' independent experiments is show one-sample t-test (C (D) fluorescence microscopy of Candida albicans stained with control IgG or mTLR2-Fc anti-Fc-Alexa594, together with ConA-Alexa488 and CFW (n=4 each). Scale bar = 5 μm I one representative of 'n' independent experiments is show (E) Docking of chitin 10 (magenta) into the TLR2 (orange) Pam2 (green) lipopeptide binding pocket (close-up, pdb: 2z7x (F) overlayed with SSL3 from a SSL3-inhibited TLR2 complex (pdb: 5d3i)  (g) MST analysis of Alexa647-labeled C10-15 and mTLR2-Fc protein with SSL3 titration (n=2) Data information I one representative of 'n' independent experiments is show (H) NF-κB activation in TLR2-transfected HEK293T cells (n=3 Data information I one representative of 'n' independent experiments is shown (for asmean+SD) ), Student's t-tes IL-8 release from primary PMNs (n=2) without or with SSL3 Data information I one representative of 'n' independent experiments is show asmean+SD) Student's t-tes  (J) BALF TNF in C57BL/6 mice (n=5/group) upon C10-15 administration without or with SSL3 Data information: Quantificatio -panel represent data (mean+SE combined from 'n' biological replicate mic one-way ANOVA with Dunnett's multiple comparison (J)  (K) ROS production between 0 and 45 min post chitin application in A. thaliana leaf pieces (n=6) Data information: Quantificatio panels ( represent dat mean±SD in K combined from 'n' biological replicate plant leave  (L) MST analysis of Alexa647-labeled C10-15 and mTLR2-Fc Protein with C5 titration (n=2) Data informatio I one representative of 'n' independent experiments is show
UBE2SHTH interacts with the donor Ub, suggesting that APC2 can prevent this conformation to facilitate the "closed" conformation.Figure 5A Molecular dynamics simulation of UBE2S (green)~Ub (yellow) reveals electrostatic interactions between the positive surface of Ub and UBE2S E139 (cutout). Rosetta model of APC2 (gray mesh, from PBD: 5L9T) docked on UBE2S~Ub would sterically clash with this extended conformation (Brown et al., 2016). B Reducing the positive charge of *Ub reduces the autoubiquitination of the E139R-substituted UBE2S variant, monitored by fluorescent scanning of an SDS-PAGE gel. C HSQC spectra displaying chemical shift perturbations and linewidth changes in 15N-labeled UBE2SUBC WT apo (black) or in complex with Ub (red) and UBE2SUBC ER/ER apo (blue) or in complex with Ub (green). D Plot of 15N-labeled UBE2SUBC WT and ER/ER 1H linewidth changes in the presence of Ub revealing the ER/ER variant has a more dynamic active-site cysteine and HTH, but a more stabilized crossover helix, indicative of the "closed" state. Shown are the best-fit values and standard deviations (SD) estimated from the covariance matrix analysis of NMR spectra with n = 2048 measurement points for 16 ppm in the 1H dimension. E Structural view of UBE2S C118 interacting with I44 of Ub in the "closed" conformation of UBE2S~Ub. UBE2S (PDB: 1ZDN) was aligned to a "closed" form of UBE2D~Ub (PDB: 4AUQ) (Dou et al., 2012; Sheng et al., 2012). F C118A substitutions decrease UBE2S autoubiquitination, measured by fluorescent scanning of SDS-PAGE gels, indicating the significance of the "closed" formation of the UBE2S~Ub for activity. G Rosetta modeling of UBE2S~Ub, in the absence of APC2, reveals multiple "open" conformations that would sterically clash with a docked Rosetta model of the APC2 (gray mesh, PDB: 5L9T) (Brown et al., 2016). Cutout showing multiple possible interactions between UBE2S and Ub. H Previous UBE2S~Ub crystal structure would also be prevented by the binding of APC2 (gray mesh, PDB: 5L9T) (Brown et al., 2016; Lorenz et al., 2016).
Figure 7A. Uptake of the uncomplexed Cy5-labeled 2'-O-MOE-PSoligonucleotide (80 mg/kg injected via the tail vein) by normal extracranial tissues was examined by fluorescence microscopy24 h after injections. B-D. Systemic treatment of intracranialGBM8tumors with uncomplexed 2'-O-MOE-PSmiR-10b inhibitor (miR-10b-i) or non-targeting control oligonucleotide (at 80 mg/kg) was not associated with toxic effects.B. No significant difference in average mice weight was observed between the anti-miR-10b and control treatment groups.C. No significant difference in average organs' weight was observed between the anti-miR-10b and control treatment groups.D. No significant difference in tissue histology using Hematoxylin and Eosin staining was observed between the anti-miR-10b and control treatment groups.
Generation of viral cDNA clones and recovery of recombinant MERS-CoV and MERS-CoV-GFP.Figure 2.Generation of viral cDNA clones and recovery of recombinant MERS-CoV and MERS-CoV-GFP.a, Schematic representation of MERS-CoV (upper panel) and MERS-CoV-GFP (lower panel) genome organization with 8 and 10 viral subgenomic overlapping cDNA fragments, respectively, used for TAR cloning. pT7, T7 RNA polymerase promoter; UTR, untranslated region; An, poly (A) tail; J represents assembly junction between two overlapping DNA fragments. Rescue of recombinant MERS-CoV and MERS-CoV-GFP. Following delivery of viral RNAs into BHK-21 cells via electroporation the cells were co-cultivated with VeroB4 cells and supernatants containing recombinant viruses produced were used to infect new VeroB4 cells. Infected cells were visualised by bright field microscopy for recombinant MERS-CoV (upper panel; 5 days post infection), and for GFP expression for recombinant MERS-CoV-GFP (lower panel, 3 days post infection). Mock represents VeroB4 cells inoculated with supernatant from BHK-21 cells electroporated without viral RNAs. Replication kinetics of the MERS-CoV-EMC (laboratory adapted isolate), rMERS-CoV and rMERS-CoV-GFP. VeroB4 cells were infected at an MOI of 0.01. Cell culture supernatants were harvested at indicated time points post infection and titrated by TCID50 assay. Data are representative of three independent experiments with two replicates per virus in each experiment. Error bars show standard deviation (SD) from the mean. TCID50/ml, 50% tissue culture infectious dose per millilitre.
Figure 3A qRT-PCR quantification of TINCR RNA expression in MMC70 and WM902B cell lines, independently infected with two shRNA targeting TINCR expression (shTINCR-1 and -2). Cells transduced with shScamble (shSCR) were used as control. TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) measured in three independent biological experiments are shown. In both cell lines TINCR knockdown efficiency is more than 70% with the two shRNAs. Statistical analyses were performed using Student t -test (**: p<0.01). B In vitro migration assay of MMC70 and WM902B cells transduced with shTINCR-1, shTINCR-2 or shSCR. Relative migration (mean±sd) from three independent experiments is indicated. Statistical analyses were performed using Student t -test (***: p<0.001). C In vitro cell proliferation of shSCR or shTINCR infected MMC70 and WM902B cells assayed 72h after infection at the indicated time points using CellTiter-Glo luminescent kit (Promega). Cell growth from three biological replicates is indicated as proliferation rate relative to control cells (mean±sd). The difference between control and TINCR-silenced samples at all time points is not significant (Student t-test). D Upper panel: in vivo growth of tumors derived from intradermal injection of shTINCR-2 or shSCR WM902B cells in NSG mice. 600,000 WM902B cells per mouse were inoculated. Tumor volumes (mean±sd) were measured 8 weeks after transplantation. Results from two independent experiments (four animals per group) are shown. Statistical analysis between control and TINCR-silenced groups was performed using the Student t -test (**=p<0.01). Lower panel: qRT-PCR analysis of TINCR RNA expression in tumors derived from in vivo transplant of shSCR and shTINCR WM902B cells (8 weeks after injection). TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) from four biological replicates (animals) from both experiments are shown. E Drug-response curves showing increased resistance of shTINCR-WM902B cells after treatment with BRAF (Vemurafenib; from 10 to 10,000 nM) and MEK (Trametinib; from 4 to 2,000 nM) inhibitors. The shSCR- (control) and shTINCR-WM902B cells were treated with increasing concentrations of drugs and their viability assayed after 72 hours using CellTiter-Glo luminescent kit (Promega). Error bars represent s.e.m. of three biological replicates. The dose response curve was fit with nonlinear regression (GraphPad Prism). F Gene ontology (GO) terms enrichment analysis of the differentially expressed genes identified by RNA-seq analysis in TINCR knockdown WM902B cells. All enrichments are significant at FDR<0.05. G Western blot analysis of a panel of proteins that are specifically associated to the proliferative or invasive phenotype. Total protein lysates were extracted from shSCR- and shTINCR-WM902B cells (lane 1 and 2) or shSCR- and shTINCR-MMC70 cells (lane 3 and 4) 10 days after infection and analyzed by immunoblot assay using the indicated antibodies. Vinculin was used as loading control.
Figure 1 A Deletion of siaA, siaB, siaC, or siaD regulates biofilm formation. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down)  B The intracellular levels of c-di-GMPin PAO1, ΔsiaC, ΔsiaB and the corresponding complemented strains were detected by LC-MS/MS  E Bacterial two-hybrid assay reveals an interaction between SiaC and SiaD. The recombinant strains harboring different vectors were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). NA represents the empty vector pBT or pTRG. The strain expressing LGF2 and Gal11P was used as positive controls  F Pull down assays confirmed the interaction between SiaC and SiaD. Cell lysates of P. aeruginosa containing pMM67EH-siaD-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads  G SPR measurements of SiaC (left) or SiaCP (right) binding at varying concentrations to SiaD. SiaC specifically interacted with SiaD with a KD of 24.7nM, whereas SiaCP failed to interact with SiaB. Shown are measured binding responses (black) and curve fits to a 1:1 interaction (red). Plots are representative from two experiments with similar results. RU, response units; KD, dissociation constant  H Production of c-di-GMP by either SiaD or SiaD with SiaC at the indicated time-point was determined by HPLC
Figure 3A&B HEK293 cells integrated with HR or NHEJ reporter were transfected with the indicated siRNAs and subjected to the HR (A) or NHEJ assay (B) as described in Methods. Data presented as mean ±SD of four biological replicates, and positive cell percentages as compared with control group. Two-tailed Student's t test, **P < 0.01. NS: no significant difference. C-F Wild-type (CTRL) or BGL3 knockdown U2OS cells were subjected to micro-irradiation; 1 hr later, cells were fixed and processed for immunostaining with indicated antibodies. Representative images of BRCA1 (C), BARD1 (D), RAD51 (E), and RPA70 (F) accumulation at sites of laser-induced DNA damage are shown.
a) The vacuolar localization of GFP–Atg8p was analysed in more than 100 wild-type, ubp3Δ, bre5Δ and atg7Δ cells containing GFP–ATG8 after 10–15 h of nitrogen starvation (right panel).figf3(a) The vacuolar localization of GFP-Atg8p was analysed in more than 100 wild-type, ubp3Δ, bre5Δ and atg7Δ cells containing GFP-ATG8 after 10-15 h of nitrogen starvation (right panel). Corresponding GFP and phase contrast images of representative cells are shown in the left panel. (b) Vacuolar cleavage of GFP-Atg8p was examined at the indicated time points (hours) by anti-GFP western blotting. (c) Wild-type, ubp3Δ and bre5Δ strains containing APE1-RFP were analysed by RFP microscopy (left) and phase contrast (right) 15 h after nitrogen starvation. Scale bars, 5 μm.
a) Frequently altered genes involved in chromatin modification processes, with each molecule positioned in parallel with its modification sites in different histones or DNA (TET1 and TET2).figf3(b,c) Significant correlations between dysregulation of chromatin modification and total number of somatic mutations (b) and overall survival probability (c). Mann-Whitney test (b) and log-rank test (c) were used to calculate statistical significance. Horizontal bars, mean value; HR, hazard ratio.
Figure 5(A) Fold change in cell number per colony in 18-hour period (n=3 pairs; each biological replicate is an average of 3 different colonies). (B, C) Live imaging reveals an increased frequency of type-II divisions in SSKO culture, as quantified in (B) (n=3 pairs; each biological replicate is an average of 3 different colonies); shown in (C) are representative images from a single experiment. (D) Cell cycle analysis of cultured Bu-HFSCs from control and Ovol2 SSKO cells (n=3 pairs)
Figure 1(A m6A dot blot of the P493-6 cells treated with Tet for 0h or 72h (A) equal mRNA loading was verified by methylene blue staining. The shown data are representative of at least three independent experiments. B) m6A dot blot of Raji cells that expressed NTC or MYC shRNAs (B); equal mRNA loading was verified by methylene blue staining. The shown data are representative of at least three independent experiments. (C) Quantification of m6A abundance by HPLC-MS. *** P<0.001 as compared to corresponding high MYC group (mean ± SD, n = 3 biological replicates, Student's t‐test). (D) Metagene profiles of m6A peak distribution along a normalized transcript composed of three rescaled non-overlapping segments: 5′UTR, CDS, and 3′UTR in P493-6 cells treated with Tet for 0h or 72h. (E) Representative GO terms analysis of transcripts with significantly up-regulated m6A peaks in P493-6 cells treated with Tet for 72h. (F) IGV graph showing location of m6A peaks on representative genes. (G) m6A-RIP assay in P493-6 cells treated with Tet for 0h or 72h. HPRT1 serves as negative control. *** P<0.001 as compared to corresponding high MYC group, ns, not significant (mean ± SD, n = 3 biological replicates, Student's t‐test). (H Western blot analysis for protein levels in P493-6 cells treated with Tet for 0h, 24h, 48h and 72h (H) HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments. I) Western blot analysis for protein levels in Raji cells that expressed NTC or MYC shRNAs (I). HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.
Accumulation of oskar mRNPs around the NE.Figure 6(A-C) Localization of EmGFP-Tm1-I (A) oskar mRNA (B) and Khc (C) around the nurse cell nuclear envelope (magenta, WGA staining).(D-F) Mean distribution profile of Khc (D) mKate2-Tm1-I, EmGFP-Tm1-I (E) and oskar mRNA (F) around the NE of nurse cells (marked with a dotted vertical line). Genotypes are indicated as follows: wild-type control with solid black line (D-F), oskar RNA null with solid grey line (D, E), Tm1eg9/Tm1eg9 with dotted black line (D, F) Lines indicate mean and 95% confidence intervals. We analysed the accumulation of mKate2-Tm1-I instead of EmGFP-Tm1-I due to unexpected GFP expression from the oskar0 allele. (G) Khc accumulation around the NE. To calculate accumulation the signal intensity measured at the position of the peak observed in the wild-type control (D, arrowhead, 356±17.6 nm away from NE) was divided by the signal intensity 2 s.d. away (d, arrow, at 356+2*410 nm). P values of pairwise Mann-Whitney U tests against wild-type control or Tm1gs rescued with EmGFP-Tm1-I are indicated above the boxplots. Numbers indicate the number of nuclei and the number of egg-chambers (in brackets) analysed.(H-K) Khc accumulation around the NE of nurse cells over-expressing EmGFP-Tm1-I within Tm1eg9/Tm1eg9 (H), and oskar RNA null egg-chambers (I) expressing either the oskar 3' UTR (J) or oskar Δi(1,2,3) (K).
SILAC‐based quantitative analysis of cell surface proteins in interphase and mitosisHistogram of the isotope ratio distribution before (left) and after (right) normalization and centering.Figure 2A Histogram of the isotope ratio distribution before (left) and after (right) normalization and centering. The x‐axis shows the mitosis/interphase ratio on a log2 scale. Note that more cell surface proteins were recovered from the interphase cells prior to normalization. B Western blot confirmation of the mitosis‐selective cell surface localization of PCDH7. Western blotting analysis of cell surface protein preparation from mitosis and interphase cells. The whole‐cell lysate and elution from streptavidin beads were blotted against TFRC, EGFR, actin, and phospho‐Histone H3. TFRC protein was enriched in interphase cell surface preparation in comparison with mitosis preparation, while PCDH7 protein was enriched in mitotic cell surface preparation. C Western blot confirmation of the mitosis‐selective cell surface localization of PCDH1. Whole‐cell lysate samples were subjected to SDS-PAGE and blotted with the anti‐PCDH1, anti‐β‐actin, and anti‐phospho‐Histone H3 antibodies, as indicated on the left. Cell surface fraction samples were blotted with the anti‐PCDH1 and anti‐ EGFR antibodies.