EMBO/sd-character-level-ner huggingface dataset:
Figure 4(A) Immunolocalization of Arp2 in a wound-healing assay. A549 cells expressing control or WDR63 were serum-starved for 6 h before they were scratched. Cells were then treated with or without 10% FBS for 30 min, followed by immunofluorescence assay. The branched actin networks were shown by immunostaining with anti-Cortactin antibody. Data shown represent three independent experiments. Scale bar: 20 μm. The lamellipodia formation was quantified by measuring the length of the outer margin of lamellipodia in individual cell (n=20 for each condition), and expressed as the proportion of the total length of the cell perimeter. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; One-way ANOVA. (B and C) Effects of full-length WDR63 (B) and WDR63 (1-200) (C) on Arp2/3-mediated actin polymerization were examined by pyrene actin polymerization assays. Various concentrations of WDR63 (B) or WDR63 (1-200) (C) were incubated with 2 μM actin (50% pyrene-labeled), 10 nM Arp2/3 and 400 nM GST-VCA as indicated. Data shown represent three independent experiments. (D) Effect of WDR63 on Arp2/3-mediated branched actin polymerization was analyzed by electron microscopy. 0.6 μM WDR63 was incubated with 2 μM actin, 10 nM Arp2/3 and 400 nM GST-VCA as indicated. Actin structures were then stabilized with addition of 2μM phalloidin, followed by electron microscopy analysis. The arrows indicate actin branch junctions. The percentage of branched actin filaments was expressed as the ratio of the number of actin filaments with branch junctions to the total numbers of actin filaments. For each condition, over 150 actin filaments were counted. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test. Scale bar: 200 nm. (E and F) Increasing amounts of purified Flag-WDR63 (0.1 μM, 0.2 μM, and 0.4 μM) (E) or Flag-WDR63 (1-200) (0.25 μM, 0.5 μM, and 1 μM) (F) was incubated with the Arp2/3 complex (10 nM) and GST-VCA as indicated in vitro, followed by GST pull-down assay. Data shown represent three independent experiments.
Cyclin D1b cooperates with h-Ras to drive tumorigenesis.Figure 4Passage-matched Ccnd1+/+ or Ccnd1KI/KI MAF lines were infected with lentivirus containing vector control or h-Ras constructs. Cells were selected with puromycin for 14 days and assayed for β-galactosidase activity. DAPI serves as a control for cell number. Images were taken at 200× magnification with insets taken at 400× magnification. The indicated cell lines were grown in complete media and assayed for h-Ras expression via immunoblot. Lamin B serves as a loading control. Cells were plated in soft agar and allowed to grow for a period of 3 weeks. Plates were then washed, fixed, and stained with 0.01% crystal violet. Colonies greater than 50 μm were counted and are plotted (top). Representative images of colony growth are shown for each cell line. Images were taken at 40× with insets taken at 200× magnification. Indicated MAF lines were injected subcutaneously into the flanks of nude mice as in (A), and tumor incidence is plotted as % of tumor-free mice over time (n = 10). Indicated tumors were harvested, fixed, and stained for the proliferative marker mKi67. Three random fields from each tumor were counted for mKi67 positivity, and are plotted as a % of total cell number, n = 5 (right: representative images). Images were taken at 400× magnification.
Figure 1Snapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across (A) a 25 kb region of chromosome 3 containing several tRNA and 5S rRNA genes Snapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across the U6 snRNA snu6 Snapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across the srp7 loci. Comparison of the distribution of ChIP-seq reads across the indicated categories of genes for Sen1 (this study) and Seb1 (data from (Lemay et al, 2016)). Genome-wide pairwise Pearson correlation coefficient matrix at a resolution of 10 bp. ChIP-qPCR analysis of Flag-tagged Sen1 at the indicated loci in a population of cycling cells (mean ± standard deviation from 4 biological replicates). ChIP-qPCR analysis of the Flag-tagged RNAP3 subunit Rpc37 and Flag-tagged Sen1 across the SPCTRNAARG.10 tRNA locus, whose TATA box was either mutated (TATA-) or not (TATA+). The mutations introduced to disrupt the putative TATA boxes are indicated in red above the graphs. The enrichment values were normalized to SPCTRNATHR.10 (mean ± standard deviation from 4 biological replicates; p-value obtained using the Wilcoxon-Mann Whitney statistical test).
Validation of select PDN drug candidates in an in vivo endotoxemia model.Figure 3 Therapeutic leads generated using PDN were directly tested for survival benefit using a murine model of endotoxemia. Select compounds were injected 24 hours before and on the day of LPS administration, using routes and doses specified in the methods. C57bl/6 female mice were injected with a high-lethality dose of E. coli LPS (38-40 μg/g) followed by a subcutaneous injection of sterile saline. Significant differences in concentration between drug and vehicle-treated pre- and post-pubertal mice are labeled with **** (p<0.0001), *** (p<0.001, ** (p<0.01), or * (p<0.05). Percent survival was compared using a log rank Mantel Cox test
Figure 3Ca2+-current-voltage relationships in response to 10 ms step-depolarizations. (A') The peak of Ca2+-influx was significantly reduced in endophilin 1-SKO (**p = 0.0024) and 1/3-DKO (***p < 0.0001) when compared to Wt. (Wt n = 30/ N = 20; 1-SKO n = 15/ N = 9; 1/3-DKO n = 39/ N = 20; Non-parametric K-W with post-hoc Dunn's correction). Quantification of CtBP2-labeled ribbons revealed a number of approximately 14-15 ribbon synapses per IHC in all groups, that remained comparable across all genotypes (Wt n = 12/ N = 9; 1-SKO n = 6/ N = 4; 1/3-DKO n = 8/ N = 6; one-way ANOVA, p = 0.717). Quantification of the cross-sectional area of CaV1.3 immunofluorescence revealed approx. 34 % smaller clusters in 1-SKOs and 24 % smaller clusters in 1/3-DKOs. (Wt n = 153/ N = 2; 1-SKO n = 102/ N = 2; 1/3-DKO n = 441/ N = 4; ***p < 0.0001; K-W and Dunn's post-hoc test). Representative maximum projections of confocal sections from organs of Corti of p15 Wt, 1-SKO, and 1/3-DKO mice stained for CtBP2 to label synaptic ribbons. Please note that CtBP2 expression is also found in the nucleus (nuclei are highlighted by dashed circles in the individual panels). Scale bars: 10 µm. 2D STED images of IHC ribbon synapses stained for CtBP2 (magenta) and CaV1.3 (green). CaV1.3 Ca2+-channels remain clustered at AZs of 1-SKO and 1/3-DKO IHCs. Scale bars: 1 µm.
Figure 7(A) Growth test of Ykt6 wild-type and mutants. ykt6∆ cells carrying a URA3 plasmid coding for wild-type Ykt6 and pRS413-GAL1pr-YKT6-GFP (wild-type or mutant) were spotted as serial dilutions on SGC-HIS to select for the mutant plasmid or 5-Fluoroorotic acid (5-FOA) to force the loss of the plasmid coding for wild-type Ykt6. (B) Effect of Ykt6 phospho-mutants on autophagy. ykt6∆ cells expressing wild-type Ykt6 plus a pRS413 plasmid encoding GAL1pr-YKT6 (wild-type or mutants with S to A mutations [SA; S182, S183A], or S to D mutations [SD; S182D, S183D]), and a plasmid expressing GFP-Atg8 were grown in SGC and then shifted to SG-N for 2 h. Cells were analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. (C) Quantification of Atg8 dots per cell from images in (B). Cells (n≥100) and Atg8 dots (n≥50) were quantified. (D) Effect of Atg1-Atg13 subcomplex on autophagosome-vacuole fusion in vitro. The vam3∆ strain carrying pRS413 plasmid encoding GAL1pr-YKT6 (wild-type or mutant), and expressing Atg9-3xFLAG and GFP-Atg8, were starved for 3 h at 30°C before purifying autophagosomes. Vacuoles were isolated from pep4∆ cells expressing Vac8-3xmCherry and then incubated with autophagosomes in the presence or absence of 50 nM Atg1-Atg13 subcomplex at 26°C for 1 h with or without ATP (E) Quantification of (D). Error bars represent standard deviation of 3 independent experiments. Fluorescence intensity of GFP-Atg8 in the vacuolar lumen was quantified by image J using the ROI (region of interest) manager tool. Vacuoles (n≥40) for each experiment were quantified. (F) Working model for the recruitment and regulation of Ykt6 during autophagy. Ykt6 arrives either after prenylation of via COPI-coated vesicles at the ER, where it binds the Dsl1 complex. Ykt6 is packaged into COPII-coated vesicles, which deliver Ykt6 independently from Atg9 vesicles to the PAS. Atg1-Atg13 kinase controls Ykt6 activity until autophagosomes are completely closed.
Figure 6(A) Confocal fluorescence images of slow skeletal muscle of 1 dpf larvae, stained with phalloidin-rhodamine to detect actin. Puncta of filamentous actin are indicated with white arrowheads, misshapen myofibers are indicated with white arrows and outlined. (B) Confocal microscope fluorescence images of slow skeletal muscle of 3 dpf larvae, stained with phalloidin-rhodamine. Puncta of filamentous actin are indicated with white arrowheads, distorted myofibers are indicated with arrows and outlined, and frayed myofibers are indicated with yellow arrowhead. (C) Confocal fluorescence images of slow skeletal muscle of 1dpf larvae, stained with phalloidin-rhodamine (red), anti-α-actinin antibodies (green), and DAPI (blue). (D) Confocal fluorescence images of slow skeletal muscle of 3dpf larvae, stained with phalloidin-rhodamine (red), anti-α-actinin antibodies (green), and DAPI (blue). Bundle of actin ringed with α-actinin indicated with white-bordered black arrowhead. (E) Myoseptal intervals of slow skeletal muscle at 1 dpf in smyhc1+/+ (n = 54), smyhc1-/- (n = 31), smyhc1R673H/+ (n = 45), and smyhc1R673H/R673H (n = 49). Distance between myosepta was measured perpendicular to rostral-caudal body axis at defined mid-body regions in phalloidin stained slow skeletal muscle fluorescence images. The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range. (F) Myoseptal intervals of slow skeletal muscle at 3 dpf in smyhc1+/+ (n = 51), smyhc1-/- (n = 32), smyhc1R673H/+ (n = 64), and smyhc1R673H/R673H (n = 38). The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range. (G) Z-disc intervals (sarcomere length) of slow skeletal muscle at 1 dpf in smyhc1+/+ (n = 380), smyhc1R673H/+ (n = 293), and smyhc1R673H/R673H (n = 268). Distance between z-discs was measured in anti-α-actinin stained slow skeletal muscle fluorescence images. The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range. (H) Z-disc intervals (sarcomere length) of slow skeletal muscle at 3 dpf in smyhc1+/+ (n = 249), smyhc1-/- (n = 278), smyhc1R673H/+ (n = 246), and smyhc1R673H/R673H (n = 252). The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.
Ubiquilins protect against starvation‐induced cell death.Figure 1(A) HeLa cells expressing GFP‐tagged UBQLN1 or UBQLN2 (or GFP for controls) were transferred to starvation medium (PBS) for the indicated time. Where specified, only serum was removed from the culture medium. Viability of transfected cells was assessed by PI staining and analysed by flow cytometry. The graph represents the mean of three experiments±s.e. (B) HeLa cells were depleted of UBQLN1 or UBQLN2 by siRNA transfection; depletion was verified by Western blot. Cells were starved for increasing periods of time, and their viability was analysed as in (A). The graph represents the mean of three experiments±s.e; *P0.05 between control and UBQLN2‐KD at 6 h (paired Student's t‐test). GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; GFP, green fluorescent protein; KD, knockdown; PI, propidium iodide; siRNA, short interfering RNA; UBQLN, ubiquilin.
a) Highly purified mTOR was incubated with radiolabelled ATP without substrate, with purified FLAG–AMBRA1 and purified FLAG–AMBRA1S52A and analysed by autoradiography.figf7(a) Highly purified mTOR was incubated with radiolabelled ATP without substrate, with purified FLAG-AMBRA1 and purified FLAG-AMBRA1S52A and analysed by autoradiography. The lower panels show a FLAG and mTOR western blot (WB). (b) HEK293 cells expressing FLAG-RAPTOR were subjected to FLAG immunoprecipitation. Protein complexes were analysed by western blotting using anti-FLAG and anti-AMBRA1 antibodies. (c) HEK293 cells were treated with Torin1 for 3 h or cultured in starvation medium for 2 h and protein extracts were analysed by western blotting using anti-pSer-52 and anti-AMBRA1 antibodies. (d) HeLa cells stably interfered for AMBRA1 were transduced with the indicated lentiviral vectors encoding for β-galactosidase, AMBRA1WT or AMBRA1S52A respectively and some of them were treated with chloroquine for 1 h. Protein extracts were analysed by western blotting using anti-LC3, anti-p62 and anti-actin antibodies. Quantification of LC3II and p62 bands is shown below. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.038. (e) GFP-LC3 MEF cells from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos were transduced with indicated lentiviral vectors and quantification of LC3 puncta per cell is shown on the right. Bars represent mean±s.e.m. of triplicate samples and 50 cells analysed per sample. n = 3 independent experiments. Significance is P = 0.001 and P = 0.003. Scale bar, 10 μm (f) HeLa cells stably interfered for AMBRA1 were co-transfected with vectors encoding Ub-Lys-63-HA and AMBRA1WT or AMBRA1S52A, respectively. Protein extracts were immunoprecipitated using an anti-ULK1 antibody and ubiquitin Lys-63-HA, ULK1, AMBRA1 and actin were analysed by western blotting. (g) HeLa cells stably interfered for AMBRA1 were transduced as in d; protein extracts were analysed by western blotting using anti-AMBRA1, anti-ATG13, anti-pATG13 and anti-actin antibodies. Quantification of the p-ATG13 band is shown below. (h) HeLa cells transfected with ULK1-HA, ULK1-Myc and different AMBRA1 constructs, respectively. Protein complexes were immunoprecipitated using anti-HA antibody and analysed by western blotting using anti-HA, anti-Myc and anti-AMBRA1 antibodies.
Chronic treatment with TKI alters brain amyloid level*Significantly different to control, Mean ± SEM, ANOVA with Neumann Keuls multiple comparison, p < 0.Figure 3Graphs represent ELISA of human Aβ1-42 in (A) brain levels and (B) bloodAβ1-42 levels in 8-month old Tg-APPmice injected I.P. every other day for 6 weeks with Bosutinib or Nilotinib (N = 10). ELISA of human soluble and insoluble brain (C) Aβ1-42 and (D) Aβ1-40 levels in 8-month old Tg-APPmice injected IP daily for 3 weeks with 5 mg/kg Bosutinib (N = 9). ELISA of (E) mouse p-Tau levels in 8-month old Tg-APP mice injected IP daily for 3 weeks with 5 mg/kg Bosutinib (N = 9). WB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) show total Tau (1st blot), serine 396 p-Tau (2nd blot), threonine 231 (AT180, 3rd blot) and serine 199 p-Tau (AT8, 4th blot) relative to actin (N = 7).
Figure 1A, B Representative bright field images of TNTs in H1299 (A) and A549 (B) cells (black arrow). These TNTs were actin-enriched, as shown by phalloidin staining (red) and not attached to the substrate (Y-Z axes). Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue). Boxed areas are enlarged and shown in panels on the right. C, D Two types of TNTs were detected in H1299 (C) and A549 (D) cells, Type 1, containing F-actin with little or no detectable microtubules (white box) and Type2, containing both microtubules and F-actin (yellow box). Anti-β-tubulin antibody (green) and phalloidin (red) were used for fluorescent staining. Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue). Boxed areas are enlarged and shown in panels on the right.
Figure 1Expression of the bacterial transcriptional repressor CggR at constant levels, i.e. independent of growth rate and substrates. Binding of CggR as a dimer of dimers to the operator (CggRO) of the synthetic cis-regulatory region, forming the CggR-DNA complex repressing transcription. At high glycolytic fluxes, fructose-1,6-bisphosphate (FBP) levels are high and FBP binds to CggR disrupting the dimer-dimer contacts, which induces a conformational change of the repressor, such that transcription of the reporter gene (YFP) can occur. The binding of FBP to CggR and consequent transcription is dependent on the FBP concentration, which correlates with glycolytic flux. The activity of the glycolytic flux biosensor is measured by quantifying YFP expression. YFP expression levels are normalized through a second reporter, mCherry, under the control of TEF1 mutant 8 promoter (PTEFmut8), to control for global variation in protein expression activity.
Figure 6(A) Schematic overview of HIV-1 infection and Tetanus toxoid (TT) stimulation of tonsillar histocultures. (B) Virus replication as assessed by quantification of RT activity in the cell culture supernatant. Shown are mean values with SD from 6 donors. (C) Production of IgG antibodies against TT from infected blocks at indicated time point. Each dot represents one donor, bar indicates mean value. Tonsillar blocks were infected with indicated virus for 24h. After washing off the virus, blocks were stimulated with TT antigen for 24h. Samples were collected at different time intervals to estimate viral replication and antibody production.
Lineage B entry into cells with known CoV receptorsa, Schematic of known coronavirus RBDs and their receptors.Figure 3:Schematic of known coronavirus RBDs and their receptors. Pseudotyped particles were either left untreated and treated with trypsin and subsequently used to infect BHK cells transfected with the coronavirus receptor indicated. Shown are data from 3 replicates. Expression and pseudotype incorporation of SARS-S-2019-nCoV RBD chimeras. Pseudotypes were used to infect cells expressing hACE2, hDPP4, hAPN, or empty vector, without protease treatment.
Fig.Figure 1A - F' Upper panels (A - F) show morphology of c4da neurons of the indicated genotypes at the third instar larval stage, lower panels (A' - F') show c4da neuron morphology at 18 h after puparium formation (APF). Neurons were labeled by CD8::GFP expression under ppk-GAL4 or by tdtomato expression in MARCM clones. (A, A') C4da neurons expressing control Orco dsRNA. Scale bars are 100 μm in A (for larval images) and 50 μm in A' (for pupal images). (B, B') C4da neurons expressing PP2A-29B dsRNA. (C, C') Homozygous PP2A-29BGE16781 mutant c4da neuron MARCM clones. (D, D') Homozygous PP2A-29BGE16781 mutant c4da neuron MARCM clones expressing UAS-HAPP2A-29B. (E, E') C4da neurons expressing dominant-negative Mts. (F, F') Homozygous mtss5286 mutant c4da neuron MARCM clones. G Penetrance of pruning defects at 18 h APF in A' - E'. ** P<0.005, *** P<0.0005, Fisher's exact test. Number of neurons for each genotype are given in the figure, the number of animals is given below in parentheses. H Severity of pruning defects at 18 h APF in A' - E' as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., ** P<0.005, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.
The UBA domain of ubiquilin is required for interaction with autophagosomes and a protective effect against starvation.Figure 4(A) Generation of Myc‐tagged UBQLN1 deletion mutants. (B) Each mutant was co‐transfected with GFP‐tagged microtubule‐associated protein 1 light chain 3 (GFP‐LC3) and the localization of each protein was analysed by microscopy. (C) Starvation‐induced cell death was assessed in cells expressing UBQLN1 or UBQLN1ΔUBA. The graph represents the mean of three experiments±s.e., with *P⩽0.05 (P=0.0017 by one‐way ANOVA test, comparing control, UBQLN1 and UBQLN1ΔUBA). No difference was found between control and UBQLN1ΔUBA at any time point. ANOVA, analysis of variance; GFP, green fluorescent protein; Pl, propidium iodide; UBA, ubiquitin‐associated; UBQLN, ubiquilin.
a) Colocalization of SLAM and bacteria in RAW264.figf5(a) Colocalization of SLAM and bacteria in RAW264.7 macrophages transfected with SLAM-mCherry and allowed to phagocytose E. coli-eGFP or S. aureus-eGFP for 0-120 min (time (in min), bottom right corners at left). Original magnification, ×60. Right, quantification of fluorescence at left. Data are representative of two independent experiments with a minimum of 40 bacteria per time point (error bars, s.d.). (b) Immunoblot analysis of phagosome isolates from RAW264.7 macrophages transiently transfected with cDNA encoding Myc-tagged SLAM (Slamf1-Myc) or mock transfected and allowed to phagocytose beads coated with E. coli outer membrane extract for 60 or 120 min. kDa, kilodaltons. Data are representative of at least three independent experiments.
Figure 6(A, B) Dendritic spine density in DiOlistically labeled DG granule cells is similar in WT and crmp2-/- mice at P30. In adults, however, spine density in crmp2-/- granule cells is increased comparing to WT. Scale bars: 5 μm. (C) Quantification of dendritic spine density in DG granule cells in the inner molecular layer (50 - 100 μm away from the soma). WT, P30: 12.5±2.2 spines/10 μm, adults: 9.95±2.3; crmp2-/-, P30: 12.86±2.1 (p>0.99), adults: 12.88±2.9 (p<0.001), mean ± SD, ***p<0.001, 2-way ANOVA with Bonferroni correction. P30: 3 animals/genotype, WT=61 dendrites, knockout=64 dendrites; adults: 3 animals/genotype, WT=41 dendrites, knockout=37 dendrites. (D, E) Analysis of branching of DiOlistically labeled DG granule cell dendrites in adult WT and crmp2-/- mice (n=3 animals, ≥25 dendrites). Quantification of granule cell branching by Sholl analysis showed no significant differences (p>0.99). Scale bars: 50 μm, mean ± SEM, 2-way ANOVA with Bonferroni correction. (F) Defects in synapse elimination in the inner molecular layer revealed by double immunostaining with PSD95 and VGluT2 antibodies (n=5 mice/genotype). GL indicates granule cell layer, IML/OML indicate inner/outer molecular layer, respectively. Density of colocalized PSD95/VGLuT2 puncta was counted. IML: WT 109±21, crmp2-/- 156±15 (p=0.004). OML: WT 281±38, crmp2-/- 297±80 (p=0.7). Scale bars: 10 μm, mean ± SD, *p<0.05, t-test. (G) Spine density in DiOlistically labeled prefrontal cortex (PFC) is similar in WT and crmp2-/- mice in both juvenile and adult mice. Scale bars: 5 μm. (H) Quantification of dendritic spine density of PFC pyramidal neurons, basal dendrites (WT, juvenile: 8.63±1.44 spines/10 μm, adults: 7.3±1.4; crmp2-/-, juvenile: 9.05±1.73, p=0.97, adults: 6.98±1.53, p>0.99), mean ± SD, 2-way ANOVA with Bonferroni's multiple comparisons test. P25: n=3, ≥50 dendrites; adults: n=3, ≥50 dendrites.