EMBO/sd-character-level-ner huggingface dataset:
Human ILC2s express high level of c-Maf.Figure 1(A) Gating strategy used to identify ILCs and ILC subsets in humans (hILCs). (B) Representative histogram and summary of the percentage and of the gMFI of c-Maf expression in hILC subsets (ANOVA analysis: F(2,18)=305.4, ****p<0.0001). Data information: Each dot represents the expression level in one donor (n=7). Bars represent mean ± SEM. (C Gating strategy for cKitlow and cKithigh ILC2s (C) D) expression level (percentage and gMFI) of c-Maf in cKitlow and cKithigh ILC2s (D) (paired T tests, ****p<0.0001). Data information: Each dot represents the expression level in one donor (n=7). Bars represent mean ± SEM.
Figure 6Activity of proteasomes within the phagosomes was determined by measuring the fluorescence intensity of fluorogenic proteasome substrate incubated with latex beads from detergent-treated phagosomes treated or untreated with Epoxomicin. The fluorescence intensities were measured at 1hr intervals and values plotted represent the ratio of the signals in the absence versus the presence of Epoxomicin Intraluminal proteasome dependent degradation of phagocytosed antigen was measured by flow cytometric analysis of phagocytosed latex beads covalently conjugated with Alexa647-OVA. Latex beads were extracted by mechanical disruption of BMDC and incubated with ATP, lysosomal protease inhibitor cocktail and varying dose of Epoxomicin at 37oC for 16hrs. Post incubation latex beads were treated with detergent and analyzed by flow cytometry (B). The mean of Alexa647 fluorescence associated with the latex beads incubated with Epoxomicin were normalized to fluorescence of untreated beads from independent experiments were plotted (n=4) (C). Ubiquitination of phagosomal cargo within the phagosomes was assessed by measuring the acquisition of GFP fluorescence by phagocytosed latex beads conjugated with OVA post-detergent extraction from BMDC expressing GFP or GFP-Ub (D). The mean of fluorescence intensities of GFP on the beads from independent experiments was plotted (E).
Cytoplasmic MT nucleation in the absence of centrosome- and Golgi-mediated MT nucleation A Representative MT network of a centrosome-less akap KO cell.Figure 6Representative MT network of a centrosome-less akap KO cell. MT-regrowth experiment after NZ treatment in centrinone-treated akap KO cells. A representative confocal image of a double labelling for EB1 (green) and giantin (blue) is shown. High magnification at right shows that growing MT asters are not associated to Golgi fragments. Summary table of proteins present (+) or excluded (-) from MT-nucleating cytoplasmic structures. Centrinone-treated akap KO cells were subjected to a MT regrowth assay and double stained with the indicated antibodies. High magnification images of the boxed areas are shown as merge (top) or individual green or red labelling (middle and bottom panels). Cells lacking AKAP450 were treated with centrinone in order to induce the formation of cytoplasmic aggregates and then transfected with siRNAs specific for CDK5Rap2 (top panels) or PCNT (bottom panels). After 3-min MT regrowth, cells were fixed and stained with the indicated antibodies. MT regrowth experiments in centrinone-treated akap KO cells transfected with either scramble or PCNT siRNAs. After a 3h NZ treatment, MTs were allowed to polymerize and, at the indicated time points after washout, cells were fixed and labelled for α-tubulin (green), PCNT (red) and CAP350 (blue). MT repolymerization experiments in centrinone-treated akap KO cells transfected with either scramble or PCNT siRNAs. After NZ treatment and removal, cell were incubated for 10 min on ice and then warmed for 3 min in culture medium containing either DMSO or 30 μM gatastatin. Cells were finally fixed and triple stained with EB1 (green), PCNT (red) and CAP350 (blue) antibodies. Enlarged views of the boxed areas are presented at right. Scale bars, 5 μm.
miR-210 regulates Fe-S biogenesis, mitochondrial function, and downstream PH pathways in vivo and is necessary to induce hypoxic PH in mice.Figure 3A Schema of comparing miR-210−/− and WT mice after hypoxia + SU5416 (PH) versus normoxia + SU5416 (control). B By immunohistochemistry (IHC), ISCU1/2 was unchanged in < 100 μm pulmonary vessels of miR-210−/−mice exposed to Hyp + SU5416 (N = 6) versus Norm + SU5416 (N = 7), NS P = 0.2833. C Fe-S-dependent Complex I-specific activity was decreased in WT PH mice as compared with control (N = 7/group, left bars, ***P < 0.0001), but activity was preserved in the lungs of miR-210−/−mice in either condition (N = 8/group, right bars), NS P = 0.3693. D IHC demonstrated that pulmonary vascular GLUT1 was increased in WT PH mice (left bars, ***P = 0.0006), but GLUT1 was unchanged in miR-210−/− mice in either condition (right bars, NS P = 0.9967). E 3-nitrotyrosine (3-NT) was increased in pulmonary vessels of WT PH mice but was reduced in miR-210−/− mice in either condition (N = 6/group) (***P < 0.0001, NS P = 0.4087). F In PH conditions, endothelin-1 was decreased in miR-210−/− lung tissue compared with WT tissue (N = 5/group), **P = 0.007. G PCNA was increased in WT PH pulmonary vessels but was decreased in miR-210−/− tissue exposed to either condition (N = 5/group) (**P = 0.0096, *P = 0.0124, *P = 0.0263 for miR-210−/−). H PCNA was increased in PECAM+ endothelial cells from PH versus non-PH mice (N = 3/group, **P = 0.0052). I Unlike WT mice (black bars) demonstrating increased right ventricular systolic pressure (RVSP) after Hyp + SU5416 (N = 10) versus Norm + SU5416 (N = 11), hemodynamic dysregulation was significantly alleviated in miR-210−/− mice (white bars, N = 11) (***P < 0.0001, *P = 0.0258, **P = 0.0059). J Compared with WT controls (N = 9), miR-210−/− mice (N = 8) displayed a blunted increase of the Fulton index (RV/LV + S) under PH versus baseline conditions (expressed as a ratio of RV/LV + S under Hyp + SU5416 versus Norm + SU5416, *P = 0.031). K-M Under PH (black bars, N = 8/group) versus baseline conditions (white bars, N = 6/group), pulmonary vascular remodeling was alleviated in miR-210−/− mice, as visualized via histology (L), and confirmed by decreased % arteriolar muscularization (K, **P = 0.001, **P = 0.0045 for miR-210−/−, *P = 0.0158) and decreased vessel wall thickness (M, ***P < 0.0001, **P = 0.0086).
Figure 5(A-B) Overall survival of ER-negative metastatic breast cancer (n=226) (A) and lung adenocarcinoma (n=719) (B) patients according to MARK4 mRNA levels found in their tumors was represented using the Kaplan Meier-plotter [25]. (C) A cDNA array for 24 matched lung cancers and surrounding normal tissue was analysed for MARK4 expression. Expression levels from tumors were normalised to their respective normal control and the percentage of cases for the indicated fold expression in the tumor represented as a pie-chart. (D-F) MARK4 protein expression was determined by histoimmunochemistry in (D-E) a tissue microarray comprising 90 lung tumor and 10 normal lung samples or (F) a home-made tissue microarray comprising 100 matched cases of primary and metastatic tumors. (D-E) The percentage of positivity for MARK4 staining in lung tumors versus normal lung samples (D) or in lung tumors according to their histological subtypes (E) is represented. (F) The percentage of cases with increased, decreased or unchanged levels of MARK4 in metastatic lesions as compared to the corresponding primary tumors is represented.
Figure 3Relative Ndrg4 mRNA (A) and Ndrg4 protein expression by Western blotting (B) and immunofluorescence (C) confirms absence of Ndrg4 in enteric neurons of Ndrg4-/- mice (n=3). EIF4E is used as a loading control in (B). qRT-PCR analysis of the Ndrg4+/+ and Ndrg4-/- ENS cells used in the co-culture model showed that Ndrg4-/- ENS cultures have reduced levels of vesicle trafficking-related genes, with the expression of Rabac1 (78.3%), Nsf (12.8%), Vamp2 (49.2%), Snap25 (13.3%), Stx1a (8.0%), Rab3a (48.1%), Stx1bp (18.3%), Caly (82.1%) and Cadps (60.4%) being down-regulated in Ndrg4-/- ENS cultures compared to Ndrg4+/+ ENS cultures. Data are derived from three independent experiments and log10 transformed ± SEM, with p-values determined using a Mann-Whitney U test. Compared to Ndrg4+/+ ENS-derived medium, addition of medium derived from Ndrg4-/- ENS cultures enhances the relative growth of intestinal epithelial organoids (E, IEOs) and accelerates the formation of new crypt buds per IEO in the course of five days (F). Medium from both Ndrg4+/+ and Ndrg4-/- ENS cultures ensures IEOs progression from crypt to (entero-) sphere, budding enterosphere, enteroid and a microadenoma-like structure. After five days of culture, significantly more nonviable structures are formed upon influence of Ndrg4-/- ENS-derived medium.
Figure 3A Permanent LAD occlusion was perfomed at ZT5 or ZT13. TTC staining (white, infarct; red, vital myocardium) and quantification of infarct size normalized to the left ventricle (LV). Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0041. B Plasma troponin I levels 24 hours after MI. Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0007. C Flow cytometry analysis of dead cardiomyocytes (CD45-, zombie+) 24 hours after MI. Student`s t-test; n = 3 mice in both groups; *P = 0.0072. D Survival rates after MI and cause of death. Log-rank test; n = 87 mice in both groups; *P = 0.0006. E Myofibroblasts within infarcts were quantified by alpha smooth muscle actin (αSMA) staining as ratio between stained and total area of random fields. Student`s t-test; n = 4 mice in both groups; *P = 0.0134. F Masson´s trichome staining of fibrosis (blue, collagen; red, vital myocardium) and quantification relative to total LV (*P = 0.0095) as well as LV anterior wall thickness (*P = 0.0068) 7 days after MI. Student`s t-test; n = 6 mice for ZT5 and n = 7 for ZT13 MI. G Analysis of relative collagen type I content identified by Sirius Redstaining 7 days after MI. Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0175. H Echocardiography assessment of ejection fraction (EF), end-systolic-volume (ESV) and end-diastolic-volume (EDV). Two-way ANOVA; n = 6 mice for no MI for both groups, n = 6 for ZT5 and n = 9 for ZT13 at 72h post MI, n = 8 for both groups at 7d post MI and n = 7 for ZT5 and n = 5 for ZT13 14d post MI; ZT5 vs. ZT13: *P = 0.0001 (EF, 3d), *P = 0.0042 (EF, 7d), *P = 0.0121 (EF, 14d); *P = 0.0253 (ESV, 3d), *P = 0.0421 (ESV, 7d), *P = 0.0005 (ESV, 14d); *P = 0.0053 (EDV, 14d).
a) Densitometric analysis relative to actin of A53T α-synuclein clearance in stable inducible PC12 cell line expressing A53T α-synuclein.figf1(a) Densitometric analysis relative to actin of A53T α-synuclein clearance in stable inducible PC12 cell line expressing A53T α-synuclein. Transgene expression was induced with doxycycline for 48 h, and then switched off (by removing doxycycline), with drug (all 1 μM) or DMSO (control) treatment for 24 h. Control condition is set to 100%. Error bars show s.e.m. (b) Densitometric analysis relative to actin of soluble EGFP-HDQ74 clearance in stable inducible PC12 cell line expressing EGFP-HDQ74. Transgene expression was induced with doxycycline for 8 h, and then switched off (by removing doxycycline) with drug (all 1 μM) or DMSO (control) treatment for 96 h. Control condition is set to 100%. Error bars show s.e.m. (c) SK-N-SH cells transfected with EGFP-HDQ74 construct for 4 h were treated with drugs (all 1 μM) or DMSO (control) for 48 h post-transfection. The proportions of EGFP-positive cells with aggregates or cell death were expressed as odds ratios and the control was taken as 1. Error bars show 95% confidence interval. (d) PC12 cells were treated with clonidine, minoxidil and verapamil (all 1 μM) or DMSO (control) for 24 h. Endogenous LC3-II levels were detected with anti-LC3 antibody and quantified relative to actin. Rapamycin (0.2 μM) was positive control. Error bars show s.e.m. (e) The proportions of EGFP-positive cells with EGFP-HDQ74 aggregates in wild-type (Atg5+/+) and knockout (Atg5−/−) Atg5 MEFs, transfected with EGFP-HDQ74 for 4 h and then treated for 48 h with drugs (all 1 μM) or DMSO (control). Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant. (a) Densitometric analysis relative to actin of A53T α-synuclein clearance in stable inducible PC12 cell line expressing A53T α-synuclein. Transgene expression was induced with doxycycline for 48 h, and then switched off (by removing doxycycline), with drug (all 1 μM) or DMSO (control) treatment for 24 h. Control condition is set to 100%. Error bars show s.e.m.
TIP30 inhibits the interaction of eEF1A1 with its essential co-factor eEF1B2.Figure 4(A) Western blot analysis of GST-pulldown assays with GST or GST-eEF1A1 and purified eEF1B2-His. Purified TIP30-His was added in indicated amounts. (B) Western blot analysis of anti-Myc immunoprecipitation (IP) in NRCM co-transduced with Ad.eEF1A1-Myc and either control virus (Ad.con) or Ad.TIP30. Endogenous eEF1B2 was detected. The IP input for eEF1B2, eEF1A1-myc and TIP30 is shown below. (C) Quantification of eEF1B2 abundance after eEF1A1-myc IP under conditions shown in (B) (D) Microscopy images of NRCM after adenoviral transduction with Ad.TIP30 or control virus (Ad.Con) and stimulation with phenylephrine (PE) and subsequent Proximity Ligation Assay (PLA). Red: eEF1A1-eEF1B2 interaction; Blue: DAPI. (scale bar: 50µm). (E) Quantification of eEF1A1-eEF1B2 interaction in conditions described in (D) (F) Microscopy images of adult mouse cardiomyocytes isolated from hearts 3 days (d) after TAC or sham surgery and subsequent PLA. Red: eEF1A1-eEF1B2 interaction; Blue: DAPI (scale bar: 100µm). (G) Quantification of eEF1A1-eEF1B2 interaction in adult mouse cardiomyocytes isolated from hearts 3 days after TAC or sham surgery and subsequent PLA (N=4 to 6 mice/group). *
Figure 5Expression of Axin2, Lgr5, Lgr4, Rspo1, Rspo2, Rspo3 and Rspo4 genes was analyzed on E15, E16.5 and adult stages by RNAscope. Arrows evidence expressing cells.
Contacts of SPIN90 with the long C-terminal helix (αD) in ARPC4 are important for activation of Arp2/3 complex.Figure 4 A. Ribbon diagram of co-complex structure with residues at the SPIN90-Arp2/3 interface depicted as Cα atom spheres colored according to side chain chemistry (grey: hydrophobic, red: basic, blue: acidic). Only the ARPC4 subunit of Arp2/3 complex is shown in the zoomed in panel for clarity  B. Time courses of pyrene actin polymerization showing the influence of mutations on the ability of SPIN90(269-722) to activate Arp2/3 complex. Reactions contained 3 μM 15% pyrene labeled actin, 50 nM Bos taurus Arp2/3 complex, and 20 μM wild type or mutant SPIN90(269-722). C. Plot of maximum polymerization rate versus concentration of SPIN90(269-722) for reactions as shown in panel (B)
Figure 1(A) IFT54 has three previously undefined and conserved regions. Schematic representation of the primary structure of Chlamydomonas IFT54 is shown on the top. Three conserved regions are shown on the bottom. Abbreviations for the species and protein entries: Cr, Chlamydomonas reinhardtii (Cre11.g467739); Hs, Homo sapiens (NP_056465.2); Mm, Mus musculus (NP_082994.1); Ce, Caenorhabditis (NP_508149.1); Dr, Danio, rerio (ABW96885); Dm, Drosophila melanogaster (NP_650353); Tt, Tetrahymena thermophila (XP_001026831.1). (B) Immunoblot analysis of whole cell samples of the HA-tagged full-length (FL) IFT54, and its deletion mutants as indicated, each expressed in the ift54 null mutant; wild type (WT) and ift54 mutant cells were used as control. Anti-HA was use to visualize the transgenic proteins, antibodies to FLA8/KIF3B were used as a loading control. (C) Histogram showing ciliary length distribution in populations of ift54 mutant cells transformed with the deletion mutant constructs as indicated. Wild type (WT) cells were used as a control. (D) Representative DIC images of wild type (WT), ift54 and ift54 cells transformed with the deletion constructs as indicated. Arrows indicate cilia bulges. Scale bar, 5 μm.
a) Left, cells were fixed and stained for anti-pTyr-416-Src (green) and with DAPI (blue).figf1(a) Left, cells were fixed and stained for anti-pTyr-416-Src (green) and with DAPI (blue). Solid arrows indicate cells with active Src in adhesions; dashed arrows show cells with active Src in puncta. Scale bars, 20 μm. Right, percentage of cells with active Src localizing to intracellular structures was quantified. Data are presented as mean±s.d. and significance is P0.001 (n=3). (b) Cells were fixed and stained for anti-FAK (red) and anti-pTyr-416-Src (green). Merged and higher-magnification images of the outlined areas are also shown. Solid and dashed arrows show Src localization to adhesions or to puncta, respectively. Scale bars, 20 μm. (c) Lysates from cells were immunoblotted with anti-FAK, anti-Src, anti-Yes, anti-Fyn, anti-pTyr-416-Src and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.
The N-terminus of Cse4 interacts in vitro with Okp1/ Ame1.Figure 4Cse4(21-129, Cse4N) and Okp1/ Ame1 were expressed and purified separately from bacteria and co-incubated before separation by analytical gel filtration. Top, representative size-exclusion chromatography (SEC) chromatogram with absorbance measurements at 280 nanometers (nm) is shown. Below, image of Coomassie Blue-stained SDS-PAGE gels with fractions from principal SEC peaks of runs with Okp1/ Ame1 alone (top), Cse4N alone (middle) and Okp1/ Ame1 pre-incubated with Cse4N before SEC (bottom). Molecular weight standards are shown in kilodalton (kDa). Intersubunit and self-link map for the Cse4N/ Okp1/ Ame1 complex formed in vitro shows direct contacts of Cse4N with both Okp1 and Ame1. Self-links and intersubunit cross-links are coloured in purple and green, respectively. The ame1 mutations S59P, N159H, S275N and a C-terminal truncation after aa 273 (ame1-273*) suppressed the growth defect of cbf1∆ cse4-R37A. cbf1∆ cse4-R37A ame1∆ strains carrying the indicated plasmid-borne ame1 alleles
Figure 2A) Summary of the hPSC lines used in this study. WT, wild type healthy control; LQT1R594Q and JLNSR594Q, isogenic pair harbouring the heterozygous and the homozygous KCNQ1 c.1781G>A mutation, respectively; LQT2N996I and LQT2corr, isogenic pair in which the heterozygous c.2987A>T KCNH2 mutation is present or corrected, respectively; hESCWT and hESC-LQT2N996I, isogenic pair in which KCNH2 is wild type or the heterozygous c.2987A>T mutation was inserted, respectively; LQT1R190Q and LQT1corr, isogenic pair in which the heterozygous c.569G>A KCNQ1 mutation is present or corrected, respectively. Arrows indicates the parent line from which each genetically matched isogenic hiPSC were generated. B) Baseline QT interval (top), baseline RR interval (middle), and baseline QT interval corrected with the Bazett's formula (bottom). Bar graphs are divided by isogenic pairs (LQT1R594Q and JLNSR594Q, left; LQT2corr, LQT2N996I, hESCWT, and hESC-LQT2N996I, middle; LQT1corr and LQT1R190Q, right) and in each graph the unrelated WT is shown as a comparison. N: 17-53. * = p<0.05. The colour of the symbol indicates comparisons and relative statistical significance. C) Representative traces of IKr steady state activation in WT hiPSC-CMs in Tyrode (left), in the presence of 5 µM LUF7346 (middle) and after the application of 5 µM E4031 to selectively block IKr (right). Inset: voltage clamp protocol. D) Average I/V relationships (left) and steady-state activation curves with superimposed Boltzmann's fittings (right) under baseline conditions (black) and in the presence of 5 µM LUF7346 (red). * = p<0.05. N: 19. E) Plot of the time constants (τ) of deactivation derived from biexponential fits (left) and representative examples (right) under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). * = p<0.05. N: 17-21.
Figure 5A. Protein/RNA co-immunopurification experiments. FLAG-tagged versions of indicated proteins were expressed in S2 cells, followed by immunopurification and RNA isolation. RNA was visualized by CIP/PNK treatment using -32P-ATP followed by denaturing polyacrylamide gel electrophoresis and phosphorimaging. Star indicates non-specific contaminant. B. Overview of experimental approach to identify dmDis3l2CM-associate RNA by high-throughput sequencing. See text for details. C. Statistics of library generated from dmDis3l2CM-bound RNA upon mapping to the Drosophila melanogaster genome. rRNA, ribosomal RNA; ncRNA, non-coding RNA; tRNA, transfer RNA; snRNA, small nuclear RNA; snoRNA, small nucleolar RNA; mRNA, messenger RNA; pre-miRNA, precursor microRNA. D. Post-transcriptional modification status of RNA classes shown in C compared to total RNA, subjected to the identical cloning protocol. E. Nucleotide identity of 3′ terminal non-genome-matching additions and change in uridine-tail content for RNA classes shown in C compared to total RNA, subjected to the identical cloning protocol. Semi-transparent bars correspond to total RNA, full bars correspond to dmDis3l2CM-IP. Tail signature reports the three most abundant post-transcriptional modifications detected in the respective RNA class and cloning approach.
ELYS knockdown reduces NPC density and nuclear levels of lamin B2, Ran, and NTF2.Figure 2MCF-10AT1k.cl2 cells were transfected with control or ELYS siRNA. (A) Representative images of cells stained for lamin B2 and B1 are shown. (B) Representative images of cells stained for lamin A/C and B1 are shown. (C) For each experiment, nuclear lamin B1, B2, and A/C staining intensities were quantified for 44-159 nuclei per condition (96 nuclei on average) and normalized to the negative control. Three biological replicates, data from one representative experiment shown. (D) Representative images of cells stained for FG-Nups (mAb414) and lamin B1 are shown. (E) Representative images of cells stained for Nup133 and lamin B1 are shown. (F) For each experiment, nuclear mAb414 and Nup133 staining intensities were quantified for 31-83 nuclei per condition (60 nuclei on average) and normalized to the negative control. To measure NPC densities, confocal NE surface images were acquired for mAb414 and Nup133 stained nuclei. NPC numbers were counted per unit area for 18-50 nuclei per condition (30 nuclei on average) and normalized to the negative control. Two biological replicates, data from one representative experiment shown. MCF-10AT1k.cl2 cells were transfected with control or ELYS siRNA. Representative images of cells stained for Ran and lamin B1 are shown. (H) Representative images of cells stained for NTF2 and lamin A/C are shown. (I) Nuclear Ran and NTF2 staining intensities were quantified and normalized to the negative control. To measure the N/C staining intensity ratio of a given cell, the average fluorescence intensity of a nuclear region was divided by the average fluorescence intensity of a cytoplasmic region. For each experiment, 54-216 nuclei were quantified per condition (112 nuclei on average). Two biological replicates, data from one representative experiment shown.