EMBO/sd-character-level-ner huggingface dataset:
Figure 7A Fluorescent images of cGAS-G3BP1 droplets in the presence or absence of 10 μM EGCG (left). The partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background (right), n = 3 biological replicates. 45 μM cGAS-mCherry and 10 μM G3BP1-mEGFP were used in these assays. Data information: Representative images are shown Scale bars, 10 μm Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. NS, non-significant. B Schematic drawing of the full-length and truncations of G3BP1. C EGCG-G3BP1 interaction analysis. EGCG was pulled-down using EGCG conjugated sepharose beads. The EGCG-G3BP1 interaction was further analyzed by immunoblotting. Data information: Representative images are shown IB, immunoblotting. WCL, whole cell lysate. D Fluorescent images of DNA-induced LLPS of cGAS, with or without G3BP1, in the absence or presence of 10 μM EGCG (left). The partition coefficient was calculated as in (A), n = 3 biological replicates. 10 μM cGAS, 5 μM G3BP1 and 100 nM dsDNA were used in these assays. Data information: Representative images are shown Scale bars, 10 μm Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. NS, non-significant. E cGAMP production assays. Recombinant cGAS (10 μM) and G3BP1 (10 μM) were incubated, in the presence or absence of dsDNA (200 nM), with or without EGCG (20 μM). The produced cGAMP was quantitatively analyzed by LC-MS/MRM. n = 3 technical replicates. Data information: Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. NS, non-significant. F cGAS-G3BP1 interaction analysis. cGAS was precipitated using anti-cGAS antibodies in U937 cells treated with EGCG as indicated. IgG served as control. The cGAS-G3BP1 interaction was further analyzed by immunoblotting. Data information: Representative images are shown WCL, whole cell lysate. IP, immunoprecipitation. G cGAMP production assays. U937 cells were untreated (left) or treated (right) with EGCG (20 μM), followed by HT-DNA transfection. The produced cGAMP was quantitatively analyzed by LC-MS/MRM. ND, not detected. n = 3 technical replicates. Data information: Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. H, U937 cells were untreated (left) or treated (right) with EGCG (20 μM), followed by HT-DNA transfection. qPCR analysis of IFNB mRNA expression in cells were performed. n = 3 biological replicates. NS, non-significant. Data information: Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type. I U937 cells were untreated (left) or treated (right) with EGCG (20 μM), followed by HT-DNA transfection. ELISA analysis of secreted IFN-β (I) were performed. n = 3 biological replicates. NS, non-significant. Data information: Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. NS, non-significant.
Limited impairment of BACE1 activity in Mgat3−/− brain.Figure 7Membrane fractions from 3-week-old Mgat3+/+, Mgat3−/−, and Bace1−/− mice were immunoblotted for CHL1 (upper), contactin-2 (middle), or syntaxin 6 (lower) (n = 4-5). The graphs show means ± SEM (**P < 0.01; two-way ANOVA with post hoc Tukey-Kramer test, P = 0.006 for Mgat3+/+ versus Bace1−/−, P = 0.008 for Mgat3−/− versus Bace1−/− in CHL1/syn6, P = 0.001 for Mgat3+/+ versus Bace1−/−, P = 0.002 for Mgat3−/− versus Bace1−/− in Contactin-2/syn6). Number of pups surviving at 4 weeks following a cross between heterozygous male and female mice.
Figure 5(A-C) Kymographs showing GFP-dynein (green) on dynamic Atto647N-microtubules (magenta) in the presence of all the regulators at different Lis1 concentrations. Protein concentrations were 10 nM GFP-dynein, 20 nM pig dynactin, 20 nM EB1, 5 µM BicD2-N, and (A) either no Lis1, (B) 1 μM mCherry-Lis1, or (C) 5 μM mCherry-Lis1. Together with BicD2, Lis1 increases the number of processive dynein runs and high concentrations of Lis1 restore plus-end localisation of dynein in the presence of BicD2-N. Microtubule orientation as indicated. (D) Averaged intensity profiles of GFP-dynein at growing microtubule plus ends in the presence of EB1, dynactin and 5 µM BicD2-N (black squares, as in A), in the presence of 1 µM mCherry-Lis1 (blue circles, as in B) and 5 µM mCherry-Lis1 (red triangles, as in C). Mean values from three separate experiments per condition (with a total of at least 49 kymographs) are shown; error bars are s. e. (E) The average number of processive DDB runs per µm microtubule length for the three different Lis1 conditions (as in A, B and, C). Error bars are s. d. *p = 0.001, **p = 0.003, ***p = 0.001 (unpaired t-test). 218, 969, and 646 processive DDB events were analysed for the three conditions, respectively (from two experiments each). (F) Bar graphs showing the mean velocity and the mean run length of processive DDB events for the three Lis1 conditions (as in A, B and C). Error bars are the s.e.m. Mean velocities were 0.37 ± 0.02 µm/s (A), 0.38 ± 0.03 µm/s (B), and 0.35 ± 0.03 µm/s (C). Mean run lengths were 3.1 ± 0.4 µm (A), 3.2 ± 0.5 µm (B), and 2.9 ± 0.3 µm (C). For each condition over 300 complexes were analysed from three different data sets. (G) Histogram of spatial initiation probabilities of DDB runs in the presence of EB1 and Lis1. 381 initiation positions were determined (from two experiments). Experiments were performed at 30°C.
Figure 1A Sphere formation of GB2 cells transfected with an shRNA targeting SIRT2 was analyzed by an In Cell Analyzer 2000. Primary spheres were re-plated to evaluate secondary sphere formation. Bars indicate mean ± s.d. of 10 wells. B Knockdown of SIRT2 causes a decrease in the sphere formation capacity of GB16. (n = 3). C mRNA levels of the indicated genes in GB2, GB4 and GB16 cells infected with a lentivirus expressing an shRNA targeting SIRT2 were measured by qRT-PCR. The results were normalized with the values for GAPDH. Bars indicate mean ± s.d. (n = 3-4). The sphere formation capacity of CD133-positive and CD133-negative cells sorted by FACS directly from a tumor sample. GB17 was infected with a control (Empty) or shSIRT2-expressing (shS2 #1) lentivirus. Bars indicate mean ± s.d. of 8 wells. E The sphere formation capacity of CD133-positive and CD133-negative cells sorted by FACS directly from a tumor sample. (Left panel) GB18 was treated with AGK2 (10 μM) or DMSO. (Right panel) Secondary sphere formation of GB18 was examined in the absence of AGK2. Bars indicate mean ± s.d. of 8 wells.
Autophagic modifications in ft mutants.Figure 8(A) EM scan of a wt (left) and ftfd mutant ommatidium (right) of a ftfd clone after 14 days. Scale bar: 1 μm. High‐magnification panels (far right, top to bottom) display autophagosomes with undigested debris, damaged mitochondria and forming phagophores (arrowheads), found in ftfd mutant cells. Scale bar: 0.2 μm for zoom‐in panels. (B) Graphs of the quantification of autophagic vesicles (AV) per photoreceptor found in EM sections of 7‐day (left)‐ and 14‐day (right)‐old ftfd clones. Significant accumulation of AV is found in mutant cells (not pigmented) with respect to genotypically wt (pigmented) cells. **P0.01 and ***P0.001 in one‐tailed t‐test. N=12 pigmented cells versus 20 non‐pigmented cells for the 7‐day graph and 13 pigmented cells versus 21 non‐pigmented cells for the 14‐day graph. (C) Confocal pictures of whole‐mount retinae of a ftfd clone aged 7 days and expressing GFP∷Atg8a ubiquitously with Tub-Gal4. Red is phalloidin marking rhabdomeres, green is GFP and the clone is marked by the absence of β‐gal staining (blue). Small GFP∷Atg8a dots accumulate specifically inside ft mutant cells (arrow). (D) Confocal pictures of whole‐mount retinae of a ftfd clone aged 7 days (left) and 14 days (right). Red is phalloidin marking rhabdomeres, blue is p62 and the clone is marked by the absence of GFP staining (green). p62 starts to gather in small dots specifically inside ft mutant cells (arrow) and then accumulates massively (arrow) in the ft mutant clones as many cells degenerate.
Figure 1(A-B) Diagram of the organization of the tracheal system at the onset of terminal cell branching (A) and one hour later (B). (C-C''') Terminal cell expressing reporters for membrane (C), ER (C') and Golgi apparatus (C''). Red arrowheads: CD4::mIFP vesicles and associated markers at the tip of the cell. (D) Mean and 95% confidence interval of tube and branch length during the first hour of growth. Numbers represent the rate of growth estimated from the slope of the curve +/-SD, p=0.39, Mann-Whitney test, n=6. (E) Distance of the tip of the tube, and the leading ER and Golgi accumulations from the cell junction that defines the base of the cell. Line and shading represent mean and 95% confidence interval, n=4. (F-I) Terminal cells expressing the general membrane marker CD4::mIFP in combination with different membrane or polarity markers: PH::mCherry, a PIP2 sensor commonly used as apical plasma membrane marker (F'), an endogenously GFP-tagged Crb (G'), polarity proteins Par3::YFP (H') and Par6::mCherry (H''). The boxed region in (H''') is shown at higher magnification in (I) over 4 time points. Red arrowheads: CD4::mIFP vesicles and associated markers at the tip of the cell.
Figure 1(A) Quantification of cystine levels (nmol/mg protein) in control (CTNSWT) (n = 4), CRISPR-generated cystinotic cells (CTNS-/-)(n = 6), and patient-derived cystinotic cells (CTNSPatient) (n = 4). (B) Quantification of transcription factor EB (TFEB )-GFP nuclear translocation in CTNSWT, CTNS-/-, and CTNSPatient, respectively. Data is demonstrated as the ratio between number of the cells with nucleus-TFEB positive over the total number of TFEB-transfected cells. The ratios were then presented as a fold change compared to control cells (n = 3). (C) TFEB mRNA expression in CTNS-/- and CTNSPatient cells compared to control cells (n = 3). (D, E) representative confocal micrographs and quantification of LC3-II accumulation in CTNSWT and CTNS-/- cells in presence and absence of 25 nM bafilomycin (BafA1) for 4 hrs, respectively (n = 3). Scale bars are 20 µm. (F, G, H) Western blotting and densitometric analyses for LC3-II/LC3-I ratio and SQSTM1 protein levels in CTNSWT, CTNS-/-, and CTNSPatient cells cultured in the presence or in the absence of 25 nM BafA1 for 4 h, respectively (n = 3). (I, J) Representative confocal micrographs and quantification of DQ-BSA and BSA in CTNSWT, CTNS-/-, and CTNSPatient cells, respectively (n = 7-14 quantified images). Scale bars are 20 µm.
Figure 2(A) Co-immunoprecipitation reveals a ClC-3-ClC-4 complex. 10% of solubilized brain membranes of WT and Clcn3unc/unc mice were directly loaded on the gel (input, or first immunoprecipitated (IP) with antibodies against ClC-3 or ClC-4. Western blots were probed for ClC-3 and ClC-4. Equivalent amounts of lysates and precipitates were loaded. * unspecific band / contamination. (B) FRET experiments show homo- and heteromerization of ClC-3 and ClC-4 constructs (fused to yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)) overexpressed in COS-7 cells. Co-expressed constructs are indicated. Graph represents energy transfer efficiencies from acceptor photobleaching, depicted as bleach corrected values (subtraction of ClC-4CFP alone). Mean values ± s.e.m. N = 20 (ClC-3YFP / ClC-4CFP); 33 (ClC-3YFP / ClC-3CFP); 26 (ClC-4YFP / ClC-4CFP); 42 (ClC-3YFP / Lamp-1CFP) cells. *** p < 0.0005 (two-tailed unpaired t test) (C) Immunolabelling shows subcellular localization of hClC-4 (top, green in merge), hClC-3 (middle, green in merge) and hClC-3unc (bottom, green in merge) of transiently transfected COS-7 cells, in comparison to either PDI or Lamp-1 (both red) as marker for the ER and late endosomes/lysosomes, respectively. (D) Co-localization of hClC-3 and hClC-4 in cytoplasmic vesicles of COS-7 cells transfected with hClC-4 cDNA (hemagglutinin (HA) tagged) together with either hClC-3 or hClC-3unc. Immunostaining used antibodies against ClC-3 and HA tag (green and red in merge, respectively).
A-BRD impairs the initiation of PCa driven by Pten-loss in a model of PCa organoids A.Figure 6A. Representative bright field images of organoids derived from mouse prostate cells treated with DMSO, BAZ2-ICR (5μM), GSK2801 (5μM) or GSK126 (5μM) inhibitors followed by transduction with shRNA-Control or shRNA-Pten. B. Quantification of shRNA-Control and shRNA-Pten organoid morphology upon treatment with DMSO, EZH2 (GSK126) and BAZ2A-BRD inhibitors (BAZ2-ICR and GSK2801). C. Representative images of Ki67-immunostained mouse prostate organoid sections expressing shRNA-control and shRNA-Pten and treated with DMSO or BAZ2-ICR (5μM). D. Quantification of Ki67+ cells in mouse prostate organoids shown in C. **< 0.01, ****P < 0.0001, two-tailed Student's t test; ns, not significant. The median values are represented by a line. E. Quantification of mouse prostate organoids per field expressing shRNA-control and shRNA-Pten and treated with DMSO, GSK126 (5μM), BAZ2-ICR (5μM), or GSK2801 (5μM). Values were normalized to shRNA-control samples. Average values of two independent experiments.
Fig.Figure 3Double-immunofluorescence labeling is depicted for pDyn and NeuN (A-C) or GFAP (D-F) in the ipsilateral dentate gyrus of KA-treated and AAV-pDyn-injected mice. Enlarged view in (F) represents 15 x 30 µm. : mature Dyn B content (measured by a Dyn B specific EIA) in the dorsal hippocampus of mice treated with KA (blue symbols) or KA and AAV-pDyn (red symbols) 1.5 (open symbols; n = 6) and 6 (filled symbols; n = 3) months after vector treatment. Naïve animals were age matched to the 1.5 months after AAV group. iH stands for ipsilateral hippocampus and cH for contralateral hippocampus.* = p<0.05; ** = p<0.01; paired t-test was used for comparison of ipsi- and contralateral hippocampi. Two-way ANOVA was used to compare Dyn levels between the early and late time interval. Mature Dyn B content in the CSF of mice treated with KA (blue symbols) or KA and AAV-pDyn (red symbols) 1.5 (open symbols; n= 6) and 7 (filled symbols; n = 4) months after vector treatment. ** = p<0.01; one-way ANOVA with Dunnett post hoc test The release of fully processed, mature Dyn B under different stimulation conditions was analyzed in microdialysates collected from the hippocampus of pDyn-deficient (KO) animals two weeks after injection of AAV-pDyn. Three baseline samples (BL; 25 min each) were collected. This was followed by 25 min low-frequency stimulation (LF), 25 min baseline, and 25 min high-frequency stimulation (HF; I). The microdialysis probe (red cross) was placed in the dentate gyrus, the stimulation electrode (black cross) in the entorhinal cortex. : Dyn B was quantified by EIA in the dialysate collected during different stimulation intensities. The red line represents the detection limit of the EIA. Data represent mean ± standard error of the mean * = p<0.05; ** = p<0.01; n=4; one-way ANOVA with Tukey post hoc test.
Figure 5A. Schematic representation of the experimental protocol. B. Representative Xenopus growth cones stained with anti-Dicer and anti-Ago2. White dashed lines delineate growth cones. Scale bars: 5µm. C. Representative E13.5 mice brain cross-section (dashed red line in the schematic) comprising the optic nerve (ON) stained with anti-neurofilament and anti-HA antibodies to detect RGC axons and (FLAG-HA2)-Dicer, respectively. Note the absence of HA signal in wild type (WT) mice. The white dashed line delineates the ON. Zoom of the triple stained ON (right panel): Dicer signal is detected inside ON cells surrounding axon bundles but not in axons per se (arrowheads). n=3 independent experiments. Abbreviations: WT, wild type; HA, (FLAG-HA2)-Dicer; E13.5, embryonic day 13.5; ONH, optic nerve head; ON, optic nerve. Scale bars: 30µm. D. Representative P0 superior colliculus stratum griseum superficiale (SGS) (sagittal sectioning of P0 brains along the dashed red line in the schematic, maximal projection of 5 µm depth). Note the presence of Dicer within the SGS marked with neurofilament. n=3 independent experiments. Abbreviations: WT, wild type; P0, postnatal day 0. Scale bars: 30µm.
Figure 8(A) Phos-tag gels showing phosphorylation of purified S. cerevisiae Mtw1 complex by Hrr251-394 and Hrr251-394:Mam187-191. Asterisks denote N-terminal proteolytic cleavage products of Dsn1, with the red asterisk denoting a previously-characterized product lacking the first 171 residues (Hornung et al, 2011). (B) Model of the intact monopolin complex. Four copies of Csm1 (blue) and two of Lrs4 (green) form the core 'V'-shaped kinetochore-binding complex (Corbett et al, 2010). Each dimer of Csm1 binds one Mam1 (orange) (Corbett Harrison, 2012), which in turn recruits one copy of Hrr25 (blue/green). Thirty-one residues of Mam1 (192-222) separate the protein's Hrr25-binding and Csm1-binding domains, which when fully extended could reach as far as ~120 Å.
a–c) Flies treated with 100 μM SMER10 (a), 200 μM SMER18 (b) or 100 μM SMER28 (c) show a shift in the distribution of the number of rhabdomeres compared with flies treated with DMSO (control) alone (2 d after eclosion).figf3(a-c) Flies treated with 100 μM SMER10 (a), 200 μM SMER18 (b) or 100 μM SMER28 (c) show a shift in the distribution of the number of rhabdomeres compared with flies treated with DMSO (control) alone (2 d after eclosion). Rhabdomere counts from all three independent experiments are included. n = 600 ommatidia (SMER10), n = 1,500 ommatidia (SMER18) and n = 600 ommatidia (SMER28). Mann-Whitney test values P 0.0001 (all SMERs). Student's t-test (1-tailed) P = 0.005 (SMER10), P = 0.004 (SMER18), P = 0.03 (SMER28) comparing means of distributions from independent experiments. These SMER concentrations cause no overt toxicity to flies (see Supplementary Methods). Distributions of DMSO-treated flies may vary when SMERs are treated in different experiments at different times. For instance, an individual experiment may have lasted slightly longer or shorter and photoreceptor degeneration is progressive and time-dependent.
SseL DUB activity reduces the recruitment of ubiquitin and autophagic markers to Salmonella microcolonies.Figure 3SseL DUB activity reduces the recruitment of ubiquitin and autophagic markers to Salmonella microcolonies.Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue) for 12 h and immunolabelled for ubiquitin (Ub, red) and (A) p62 (green) or (B) LC3 (green) (false coloured, scale bars, 5 µm). The far right panels show merged images of p62 or LC3, ubiquitin and Salmonella. Arrows indicate SCV-associated ubiquitin labelling with p62 or LC3. (C-E). Quantification of the relative fluorescence intensity of (C) ubiquitin, (D) p62 and (E) LC3 per microcolony (see Materials and Methods). Cells were processed as in (A) and a minimum of 30 microcolonies analysed for each bacterial infection per experiment. Results from representative experiments showing the relative mean fluorescence intensity ± SEM are shown. Similar results were obtained in at least 3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001.
Figure 3A Schematic representation of p73 isoforms. TAD, transactivation domain; DBD, DNA-binding domain; OD, oligomerization domain; SAM, sterile alpha motif domain. B Immunoblotting analysis of p73 in GB2 cells infected with a control (Empty) or shTP73-expressing lentivirus for 96 h. C Growth curve of GB16 cells infected with the indicated lentiviruses. Bars indicate mean ± s.d. (n = 3). Ectopic expression of the ∆N isoform of p73β suppresses the expression levels of PUMA and GADD45 induced by SIRT2 knockdown in GB2 cells. Four days after lentiviral infection, the expression levels of PUMA and GADD45 mRNA were measured by qRT-PCR. Bars indicate mean ± s.d. (n = 3). E GB2 and GB4 cells [5 × 104 and 2 × 104 cells, respectively (indicated by the dashed lines)] were infected with the indicated lentiviruses. After 96 h, the number of viable cells was counted. Bars indicate mean ± s.d. (n = 4). F HEK293 cells were transfected with TAp73α along with ΔNp73β. PUMA mRNA was measured by qRT-PCR analyses. Bars indicate mean ± s.d. (n = 3). GB2 cells (5 × 104 cells, dashed line) were infected with the indicated lentiviruses, respectively. After 96 h, the number of viable cells was counted. shTP73 corresponds to both α and β isoforms of p73. Bars indicate mean ± s.d. (n = 4). H GB2 cells were transfected with TAp73α along with NLS-SIRT2 (nuclear-localizing mutant of SIRT2) or 3mut (deacetylase-inactive, nuclear-localizing mutant of SIRT2) and a reporter construct consisting of the promoter region of PUMA fused to a luciferase gene (Left panel). Reporter activities were determined by Dual luciferase assays (Right panel). Bars indicate mean ± s.d. (n = 3). I GB2 cells were electroporated with TAp73α along with NLS-SIRT2 (nuclear-localizing mutant of SIRT2) or 3mut (deacetylase-inactive, nuclear-localizing mutant of SIRT2). The expression of PUMA and GADD45 was measured by qRT-PCR analysis. Bars indicate mean ± s.d. (n = 3)
Basolateral α-β Spectrins are required for Hippo signalling and membrane tension in the Drosophila follicular epithelium.Figure 6A Control egg chamber stained for DAPI to mark nuclei.B Overexpression of Yki3SA stimulates follicle cell proliferation (MARCM clone, GFP positive).C-F Mutation of βH-spectrin/karst (C) or crb (D) does not increase follicle cell proliferation (GFP negative clone), while mutation of α-spectrin (E) or β-spectrin (F) stimulates follicle cell proliferation (GFP negative clone). G Control egg chamber stained for Cut, a marker of proliferating cells that is normally down-regulated after stage 6 of oogenesis.H-K Mutation of βH-spectrin/karst (H) or crb (I) does not increase Cut expression in follicle cells (GFP negative clone), while mutation of α-spectrin (J) or β-spectrin (K) stimulates Cut expression in posterior follicle cells (GFP negative clone).L Control egg chamber showing ex.lacZ reporter gene expression.M-P Mutation of βH-spectrin/karst (M) or crb (N) does not increase ex.lacZ expression in follicle cells (GFP negative clone), while mutation of α-spectrin (O) or β-spectrin (P) stimulates ex.lacZ expression in posterior follicle cells (GFP negative clone). Q Wild-type follicle cells showing normal hexagonal packing of epithelial membranes.R Mutation of β-spectrin disrupts the normal hexagonal packing, suggesting a role in maintaining membrane tension.S Clone of β-spectrin mutant cells (GFP negative) showing differential membrane tension at the interface with wild-type cells (GFP positive).