EMBO/sd-character-level-ner huggingface dataset:
a–c) Temporal changes in the green fluorescence pattern of photoactivated NIH3T3 cells stably expressing KFERQ–PS–CFP2 maintained in the absence (black circles) or presence (white squares) of serum for the indicated times.f2(a-c) Temporal changes in the green fluorescence pattern of photoactivated NIH3T3 cells stably expressing KFERQ-PS-CFP2 maintained in the absence (black circles) or presence (white squares) of serum for the indicated times. (a) Representative images of cells maintained in serum-free media. Quantification of the number of puncta per cell (b) and green fluorescence intensity (c). Values are expressed as percentage of fluorescence at time 0 and half-lives (t1/2) are indicated. Differences in the number of puncta and fluorescence intensity between cells maintained in the presence and absence of serum (*) were significant for P=0.048 (16 h) and P=0.04 (24 h) (t-test). (d) The same cells imaged in the cyan channel, before and at the indicated times after photoconversion (arrow) by exposure to 405 nm light. Top: Representative images of the cyan fluorescence. Bottom: Quantification of the intensity of the cyan fluorescence. Values are expressed as times the intensity before photoconversion that was given an arbitrary value of 1. Values are mean+s.e. of 3 different experiments with >10 cells counted per experiment. Scale bars, 5 μm.
Figure 4RAP1 expression in HeLa cells treated with the telomerase inhibitor BIBR1532 for 25 days (20 µM final concentration). Fifteen days before cell harvesting, doxycycline (DOX; 1 µg/µl final concentration) was added to deplete the expression of RAP1. Telomere length analysis by southern blotting of the samples described in A. The size of the main intensity peak is indicated at the bottom of the gel. Number of fusions in HeLa cells after 25 days in culture. Cells were maintained with BIBR1532 during the whole period of the experiment, while doxycycline (1 µg/µl final concentration) and the infection with shControl, shLIG3 or shLIG4 was carried out for the last 15 days of the experiment. Data represent mean +/- SD of three biological replicates are shown. Statistical analyses were performed using Mann-Whitney U test (*p < 0.05; **p < 0.001; ***p < 0.0001). Examples of metaphase spreads hybridized with a telomeric PNA probe (green) when RAP1 was depleted (+DOX). Telomerase was inhibited with BIBR1532 for 25 days. Scale bar = 10 µm. Quantification of chromosome aberrations observed in RAP1 depleted cells (+DOX) or control (-DOX). Data represent mean +/- SD of three biological replicates. Approximately 15 metaphase spreads were analyzed per replicate with a total of 2300 chromosomes examined per condition. *p < 0.05; two-tailed Student's t test.
IFT74/81 interaction analysis using different IFT22 mutants A.Figure 4A. SDS-PAGE gel of a Ni2+-NTA pulldown using His-tagged IFT74342-401/81397-450 and untagged IFT22 (WT and mutants). Pulldowns were done from cell lysates of co-expressed proteins. Lanes 1-4 show similar total expression levels of the different co-expressed constructs (input samples). Lanes 5-8 show pulldown elutions. The IFT22S19N (inactive GTPase mutant) and the IFT22A86R mutant (IFT74/81-binding mutant) did not interact with the IFT74342-401/81397-450 complex in the pull-down experiment. B. SDS-PAGE gel of a Ni2+-NTA pulldown using the His-tagged IFT7479-401/811-450 and untagged IFT22 (WT and mutants). Pulldowns were done from cell lysates of co-expressed proteins. Lanes 1-4 show similar total expression levels of the different co-expressed constructs (input samples). Lanes 5-8 show pulldown elutions. The IFT22S19N (inactive GTPase mutant) and the IFT22A86R mutant (IFT74/81-binding mutant) did not interact with the IFT7479-401/811-450 complex in the pull-down experiment. C. SDS-PAGE gel of a Ni2+-NTA pull-down of full-length IFT25/27/74/81 complex with His-tagged IFT22 (WT and S19N mutant). The nucleotide-binding deficient mutant IFT22S19N interacts with IFT25/27/74/81 in the pull-down experiment. D. SDS-PAGE gel of a Ni2+-NTA pull-down of full-length IFT25/27/74/81 complex with His-tagged IFT22 (WT and A86R mutant). The IFT74/81-binding mutant IFT22A86R fails to pull down the full-length tetrameric complex.
Fig.Figure 3(A and B) Representative images of NSCs derived from FKRP- and corrected-iPSC lines expressing SOX1, SOX2 and nestin. (C and D) Quantification of percentage of SOX1+ (C) and SOX2+ (D) cells in culture. The efficiency of neural induction is more than 99% in FKRP- and corrected-iPSC (5D17, 5D23 and 3B17) lines. Data are mean ± s.d. n = 4 technical replicates. Data information: Scale bars, 50 μm. (E and F) FKRP- and corrected NSC lines can be further differentiated to cortical neural progenitor cells, expressing PAX6, OTX2 and vimentin. (G-I) Quantification of percentage of PAX6+ (G) and OTX2+ (H) cells in culture. About 91-98% of cells derived from FKRP, 5D17, 5D23 and 3B17 NSC lines express PAX6 (G). About 93-96% of cells derived from FKRP, 5D17, 5D23 and 3B17 NSC lines express OTX2 (H). Of the OTX2+ population, about 60-67% cells are also Ki67+ cycling progenitors (I). Data are mean ± s.d. n = 4 technical replicates. Data information: Scale bars, 50 μm. (J and K) Glutamatergic projection neurons derived from FKRP and corrected (5D17, 5D23 and 3B17) progenitor cells. The vast majority of neurons contain vGlut1+ punctae in their neurites (labelled by Tuj1). Right panels are enlarged images from the insets of left panels. Data information: Scale bars, 50 μm.
Physiological characterisation of pyk1 variant.Figure 4(A) Growth measured by optical density (OD) at 600nm for 3 biological replicates each of the T- and A-strains over 6 hrs of exponential growth in YES media. The x-axis refers to the time since inoculation of the culture. (B) The same data as in (A) plotted on a log2 scale (solid lines) with lines fitted (dotted lines). The doubling time is the inverse of the slope. (C) Media samples were taken from the same cultures (n=3 for both strains) at the 8 hr timepoint and the remaining glucose was quantified. The consumed glucose was calculated based on the amount of glucose measured in the same, fresh media and normalised to the OD of each culture at the time of sampling. (D) Oxygen consumption rates in A- and T-strains (ratio of means=0.65, p=0.0002, Welch's T-test, 3 biological replicates, each measured in technical duplicates). Data information: Vertical bars show the mean of the data. Error bars and shaded areas in all cases denote standard deviation. Significance keys: * p<0.05, *** p<0.0005 (Welch's t-test). (E) Culture dry weight after 24 hrs was measured reported as a fraction of the weight of glucose put into the media for 3 biological replicates per strain. Data information: Vertical bars show the mean of the data. Error bars and shaded areas in all cases denote standard deviation. Significance keys: * p<0.05, *** p<0.0005 (Welch's t-test).
a) A HeLa cell expressing KR-dMito, EGFP-LC3B and EBFP2-parkin (cyan) were 559 nm illuminated (white dotted circle region; immediate loss of KR fluorescence).f2(a) A HeLa cell expressing KR-dMito, EGFP-LC3B and EBFP2-parkin (cyan) were 559 nm illuminated (white dotted circle region; immediate loss of KR fluorescence). (b-d) Magnified view of the photo-inactivated region in a 34-93 min after 559 nm illumination. (b) Induction of parkin-mediated mitophagy on a single, long mitochondria tubule (white arrows). (c) LC3B-coated structures were recruited onto distinct spots on the photo-impaired mitochondrial tubule (white arrows: the location of the original impaired single mitochondrial tubule). (d) White dotted line region outlined in c, LC3 structures segregated from the main parkin-labelled cluster, carrying away bits of the parkin-labelled mitochondria with them. Two individual events are labelled in the image sequence with white and yellow arrows, respectively. Scale bars (all panels), 5 μm.
Bevacizumab therapy leads to reduced vessel density and increased Ang-2 expression.Figure 6Figure 6: Bevacizumab therapy leads to reduced vessel density and increased Ang-2 expression. Immunohistochemistry stainings with antibodies directed against CD31 (A) and Ang-2 (B) in treatment-naive GBM, post-radiochemotherapy (S/RTx/CTx) and post-radiochemotherapy + bevacizumab (S/RTx/CTx/Bev) therapy are displayed. Quantitative analyses of microvessel densities and Ang-2+ vessels are shown in (C) and (D). The ratio of Ang-2+ vs. CD31+ vessels was determined in (E). Western Blot and corresponding quantification of CD31 and Ang-2 in control and aflibercept treated mice (F). CAIX staining of treatment-naive GBM and S / RTx / CTx / Bev patients (G). Analyses of hypoxic (CAIX+) area with regard to the whole tumor in human GBM (H). Tumor-infiltrating lymphocytes (CD8/CD3) in human patients (I). Kruskall-Wallis (Dunn´s post test). * p < 0.05, *** p < 0.005; treatment-naive GBM n=26, S/RTx/CTx n=7, S/RTx/CTx/Bev n=29; data are mean ± SEM
tyrosine phosphorylation in adult mice.Figure 5LSM images of a whole mount stained adult Prox1-CreERT2;Ilk+/+ mouse ear (referred to as "Adult Control") and Prox1-CreERT2;Ilk∆/∆ mouse ear (referred to as "Adult ILK K.O."), with higher magnification images indicated by dashed lines. Scale bars: 500 and LSM images of a whole-mount stained adult control and ILK K.O. ear, with higher magnification images indicated by dashed lines. Arrows point to proliferating LECs. Scale bars: 100 and 20 µm, respectively Dermal lymph vessel density as determined by VEGFR3-positive area normalised to the total analysed area of adult control or ILK K.O. mice (n = 4 control and n = 5 ILK K.O. mice), LEC proliferation as determined by phospho-Histone H3-positive LECs normalised to analysed VEGFR3-positive area in the skin of adult control or ILK K.O. mice (with n = 7 control and n = 5 ILK K.O. mice), VEGFR3 tyrosine phosphorylation as determined by ELISA of skin lysates of adult control or ILK K.O. mice (n = 3 mice per genotype) LSM images of a whole mount stained adult control and ILK K.O. cornea, with detailed images on their right (L", M"). Arrows point to lymph vessels protruding into the cornea (encircled by a dotted line). Scale bars: 500 µm and 100 µm, re Number of lymph vessels in control or ILK K.O. corneas (n = 5 and n = 7 corneas),
Figure 4(A) ACE2-Fc blocked the entry of Spike-expressing pseudotyped lentivirus into HEK293T-ACE2 and H1975-ACE2 cells. The relative luciferase activities, normalized to the only virus group, represent the efficiency of virus entry. MOI: Multiplicity of infection. (B) Confocal microscopy images of the airway organoids. The green fluorescence represents the specific staining of indicated monoclonal antibodies and Alexa Fluor 488-conjugated secondary antibodies. DAPI was used as a nuclear counterstain. Hematoxylin and eosin (H&E) stain: hematoxylin stained the nuclei in blue color; eosin stained the cytoplasm in pink color. Scale bars equal to 20 µm. (C) Lung cancer A549 cells, human normal bronchial epithelial cells (HBEpc), and HBEpc-differentiated cells (airway organoids) were harvested for immunoblotting with the indicated antibodies. (D) Blockage of pseudovirus entry into airway organoids by ACE2-Fc. Mixtures of pseudotyped lentivirus with or without ACE2-Fc were cocultured with airway organoids for 72 hr. The virus entry was determined by quantifying the luciferase activity in the cell lysates.
Figure 4Schematic diagram of miR-AB-based lentiviral (LV) and retroviral (RV) vectors. miR-AB shRNAmir expression is driven by a composite promoter composed of the endogenous 5' LTR of HIV and a eukaryotic promoter in lentiviral vectors, or by the endogenous 5' LTR of MSCV in retroviral vectors. Eight fluorescent proteins with emission peaks across whole visible spectrum are controlled by mouse PGK promoter. A woodchuck hepatitis post-transcriptional regulatory element (WPRE) is used to enhance transgene expression. The excitation and emission spectra of these fluorescent proteins are shown in FPbase Spectra Viewer (https://www.fpbase.org/spectra/). The five commonly-used lasers are indicated in the spectrum. The excitation spectrum of Azurite is shown as that of its parental protein EBFP due to lack of information in the database. Nomenclature of fifty-six miR-AB-based lentiviral and retroviral vectors with different promoters and different fluorescent protein reporters. AH promoter (human beta actin promoter(Ng et al, 1989)/human leukaemia viruses RU5 region)-based miR-AB lentiviral vectors, the most recently developed one, also exhibited strong RNAi efficiency. Puromycin was also engineered into this toolkit for antibiotic-based screening of transformants. Lentiviral vectors with GFP reporter that are controlled by the promoters used in miR-AB lentiviral vectors were constructed for easy assessment of the promoters activity on microscope or flow cytometer. CD8+ T cells were activated and then co-transduced with miR-AB retroviruses targeting CD4 with Azurite, GFP, Ametrine, mOrange, mCherry2 or E2-Crimson as fluorescent reporters. The fluorescences of all fluorescent proteins were plotted on contour plots showing the fluorescent separation of each pair of the fluorescent proteins. The data was a representative of at least three biologically independent experiments. 293T cells were co-transduced with four miR-AB lentiviruses at 1:1 ratio, which carries a neutral shRNAmir targeting CD4 or CD19, with Azurite, GFP, Ametrine or mOrange as reporter respectively. Cells were cultured for 22 days and fluorescence positive cells were quantified at the time points as indicated. Representative contour FACS plots cells on day 4 and day 22 shown (top panels). Cell numbers of untransduced, single fluorescence positive cells and quadruple positive cells were quantified and normalized to untransduced cells on day 4, and plotted over time (bottom panel). Data are shown as mean ± s.d. and n=4. NS indicates no significant difference (P>0.05, two-tailed unpaired Student's t-test).
−Vret complex (A) Immunofluorescence showing the Vret (red) and Ago3 (green) signals in DDX43-depleted BmN4 cells (siDDX43).Figure 2(A) Immunofluorescence showing the Vret (red) and Ago3 (green) signals in DDX43-depleted BmN4 cells (siDDX43). DAPI shows the nuclei (blue). Scale bar: 10 µm. The insets show high-magnification images of the part indicated sections. (B) Immunoprecipitation and western blotting showing that DDX43 associated with Vret and Ago3. (C, D) Immunoprecipitation of endogenous Siwi, DDX43, and Vret bound with Ago3 from DDX43-depleted (C) or Vret-depleted (D) cells. The factors were detected by western blotting. Ago3 interacts with Vret and DDX43 independently of the other. (E) Immunofluorescence showing Ago3 (red) and Flag-DDX43 (green) signals in BmN4 cells. DAPI shows the nuclei (blue). Arrowheads indicate colocalization of these factors. Scale bar: 10 µm.
Stable triple knockdowns of tumor suppressors p53, PTEN and RB in healthy FT organoids.Figure 3Experimental approach for genetic manipulation of FT epithelial cells (FTECs). FTECs were sequentially transduced in 2D culture with replication-deficient viruses containing specific shRNAs and different selection markers. After sorting cells were seeded in Matrigel for organoid formation. Scale bars: 2 cm (tissue), 200 µm (2D culture) and 500 µm (3D culture). Confirmation of robust knockdowns of p53, PTEN and RB in FT organoids on RNA and protein level. Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ±SEM from 3 different donors. A representative western blot from one donor is depicted. RNA as well as protein samples were taken at passage 1 or 2 of each organoid line. KD - Knockdown, Vec Ctrl - Vector Control, WT- Wild type Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis. Confocal images of triple KD organoids reveal increased DNA damage (yH2AX, marked by asterisks), atypic nuclei (DRAQ5) and loss of apicobasal polarity. Scale bar: 20µm. Confirmation of robust knockdowns of p53, PTEN and RB in FT organoids on RNA and protein level. Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ±SEM from 3 different donors. A representative western blot from one donor is depicted. RNA as well as protein samples were taken at passage 1 or 2 of each organoid line. KD - Knockdown, Vec Ctrl - Vector Control, WT- Wild type Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis. Presence of ciliated cells (arrowheads), as revealed by immunostaining against detyrosinated Tubulin (dtyrTub) in triple KD organoids, proves capacity for terminal differentiation. Scale bar: 50µm.
Figure 6A. Experimental design of MS-based quantitative phosphoproteomics analysis of wt- and T693A-expressing T47D cells stimulated with FGF10 for 40 or 60 min. B. Hierarchical clustering of the phosphorylated sites differentially quantified in wt- and T693A-expressing T47D stimulated or not with FGF10 for 40 or 60 min.. Four clusters for plasma membrane Response (PMR), Acquired Response (AR), Late Response (LR), and T693 Phosphorylation Dependent Response (PDR) are highlighted with black lines and color-coded on the right with blue, medium, or dark blue and yellow, respectively. The intensity of phosphorylated sites is presented on the logarithmic scale with intensity below and above the mean color-coded in blue and red, respectively. C. KEGG pathways enriched in each cluster. D. STRING-based and Cytoscape-visualized network of phosphorylated kinases color-coded based on clusters shown in Figure 6C. The border of known regulatory sites is coloured in burgundy. The shape depends on the known function of the regulatory site. E, F Lysates from T47D (E) and BT20 (F) depleted of EGFR, transfected with wt or T693A, and stimulated or not with either FGF10 or TGFα for the indicated time intervals were immunoblotted with the indicated antibodies.
Screening of Drosophila dsRNA library by PicoGreen nucleoid fluorescence in S2 cellsMicrographs of S2 cells stained with PicoGreen, following 5 days of treatment with the dsRNA indicated.Figure 1Micrographs of S2 cells stained with PicoGreen, following 5 days of treatment with the dsRNA indicated. GFP and tamas (Polg, CG8987) were used as negative and positive controls, respectively. In rescreening, CG5924 was also used as a positive control. Both were detected in the blinded screen as positives. Other images show a typical negative (CG31380), a typical positive (CG6413) and a typical case of a target classed as abnormal, in this case CG2028 (CkIα), which showed a decreased number of nucleoid signals per cell. Images are optimized for brightness and contrast but with no other manipulations. Scale bar indicates 50 μm.
, A role for CD22 dynamics and organization in restraining BCR signaling in naïve B cells.Figure 8Figure 8, A role for CD22 dynamics and organization in restraining BCR signaling in naïve B cells.(A) In the steady-state, CD22 dynamics and nanoclustering are regulated by CD45. Sialylated CD45 works as a spacer between mobile molecules of CD22 displaying high amount of sialic acid. This organization limits the extent of active CD22 (red tails, side view) and restrains the proximity of CD22 to the BCR (long arrow, side view). This equilibrium allows a low level of basal signalling required for B cell survival.(B) When CD45 is absent, the tendency of CD22 to form self-interactions via sialic-acid binding increases together with CD22 nanocluster size and inhibitory activity. Bigger clusters of hyperactive CD22, no longer trapped by CD45, can access the BCR more easily (short arrow, side view) restraining the signalling.(C) The inability of CD22 to bind the sialic acid (Cd22R130E) on either CD45 or neighboring CD22 molecules decreases the size of CD22 nanoclusters with a concomitant increase of CD22 lateral mobility (dashed arrows, side view and top view) and phosphorylation. In this situation CD22 is able to explore larger area and access more BCRs (short arrow, side view) thus leading to a higher signal inhibition.
Figure 5A Schedule for anti-Ly6g antibody (Ab) treatment of Hepa1-6 tumor-bearing mice. Ab was intraperitoneally injected every 3 days as indicated. PBS was injected as a mock control. B Representative flow cytometry plots of splenic G-MDSCs measured in tumor-free untreated Tet2-/- (KO) and WT mice and after anti-Ly6g or PBS injections into tumor-bearing mice. Flow cytometric analysis of T cells in Tet2-/- mice and littermate control mice (WT) with or without anti-Ly6g treatment after tumor challenge. C Tumor growth curves in WT and KO mice treated with either PBS or anti-mouse Ly6g (n=5). D Day-18 tumor weights at the endpoint of (C) [n=11, PBS (-) injected tumor-bearing WT and KO mice; n=12, anti-Ly6g antibody (+) injected tumor-bearing WT and KO mice]. E Percentages of CD8+ T cells from the spleens of tumor-bearing mice with or without anti-mouse Ly6g treatment [n=17, PBS (-) injected tumor-bearing WT and KO mice; n=6, anti-Ly6g (+) antibody injected tumor-bearing WT and KO mice]. F Percentages of CD4+ T cells from the spleens of tumor-bearing mice with or without anti-mouse Ly6g treatment [n=16, PBS (-) injected tumor-bearing WT and KO mice; n=6, anti-Ly6g (+) antibody injected tumor-bearing WT and KO mice Flow cytometric analysis of T cells in Tet2-/- mice and littermate control mice (WT) with or without anti-Ly6g treatment after tumor challenge.
Figure 3Statistical analysis of Pc and stx double mutant stxd77;; pcXT109 sleep profile compared with controls. total sleep (From left to right mean±SEM: 378.6±12.25, n=77; 455.0±11.17, n=80; 286.6±13.68, n=41; 356.7±14.50, n=43; 468.7±10.22, n=77; 467.2±13.49, n=80; 498.5±11.91, n=41; 500.9±17.84, n=43). Statistical analysis of Pc and stx double mutant stxd77;; pcXT109 sleep profile compared with controls. sleep bout duration (From left to right mean±SEM: 22.55±1.219, n=77; 40.86±3.271, n=80; 14.33±0.889, n=41; 20.84±2.209, n=43; 53.72±4.560, n=77; 64.26±6.380, n=80; 66.39±9.010, n=41; 81.38±11.49, n=43). Statistical analysis of Pc and stx double mutant stxd77;; pcXT109 sleep profile compared with controls. number of sleep bouts (From left to right mean±SEM: 19.67±0.5610, n=77; 15.91±0.6843, n=80; 21.79±0.8670, n=41; 21.20±0.9624, n=43; 12.69±0.5957, n=77; 11.58±0.5415, n=80; 13.31±0.7201, n=41; 11.34±1.001, n=43). Statistical analysis of Pc and stx double mutant stxd77;; pcXT109 sleep profile compared with controls. (D)sleep latency of stxd77;; pcXT109 (From left to right mean±SEM: 52.20±5.496, N=7; 22.76±2.621, N=9; 53.68±8.744, N=3; 31.55±7.770, N=3). Statistical analysis of Pc and stx double mutant stxd77;; pcXT109 sleep profile compared with controls. Quantitative RT-PCR of Octβ2R in adult head of stxd77 and w1118 control. (Data represent mean±SEM: w1118, 0.8853±0.1177; N=3; stxd77/Y, 0.3263±0.1575；N=3). Rescue of stxd77 by over expressing Octβ2R in ellipsoid body. From left to right mean±SEM: 399.6±11.49, n=50; 400.3±10.48, n=60; 349.9±14.77, n=48; 495.8±16.43, n=28; 470.1±13.04, n=44; 423.6±14.32, n=47; 548.6±8.707, n=50; 499.0±8.225, n=60; 490.7±16.26, n=48; 494.7±11.99, n=28; 480.2±14.64, n=44; 437.4±14.85, n=47. Rescue of stxd77 by Pc RNAi in ellipsoid body. From left to right mean±SEM: 399.9±11.88, n=48; 409.8±11.50, n=49; 348.7±20.19, n=32; 495.8±16.43, n=28; 459.3±14.78, n=61; 407.5±15.66, n=40; 550.7±8.881, n=48; 516.0±11.76, n=49; 502.3±15.08, n=32; 494.7±11.99, n=28; 546.3±11.35, n=61; 514.2±18.94, n=40. Note that the same data is used for genotype stxd77;; EB1-Gal4 in panel F and G. Colocalization of stx-Gal4 with Octβ2R-Gal4. Scale bar showed was 10um. Dam-ID experiment to identify the binding of Pc on Octβ2R genomic locus. Primers used were shown in (I). The fold enrichment of different sites were shown in (J), iab-7 was used as a positive control for Pc binding (From left to right mean±SEM: 8.9±0.3, 1.2±0.06, 2.2±0.3, 3.3±0.7, 1.7±0.3, 14±0.06, 1.2±0.3, 5.3±0.7, 1.9±0.3, 15.8±0.06, 19.2±0.3, 26.7±0.7, 15.2±0.7, N=3). Octβ2R was upregulated in PcXT109 mutant (Data represent mean±SEM: w1118, 1.004±0.0609, N=3；pcXT109/+, 1.447±0.1300, N=3).