EMBO/sd-character-level-ner huggingface dataset:
Figure 4Transmission electron microscopy of GSC#9 treated with vehicle (DMSO) or MPZ (20 μM) for 16 hours. ER: endoplasmic reticulum; MVB: multivesicular bodies; lys: lysosome; mit: mitochondria; nuc: nucleus. Red stars denote lysosomes, blue stars vacuoles. Confocal analysis of LAMP2 staining (red) at 0, 1, 2, 4, and 6 hours post-MPZ (20 μM) treatment. Nuclei (DAPI) are shown in blue. Scale bar: 10 μm. Western-blot analysis was performed in total protein lysates from GSC#9 transfected with non-silencing duplexes (sic) or MALT1 targeting siRNA duplexes (siMALT1). Alternatively, western-blot analysis of LAMP2, CTSD, and MALT1 was done in total protein lysates from GSC#9 treated for 16 hours with MPZ (20 μM) or Z-VRPR-FMK (75 µM). DMSO was used as vehicle. GAPDH served as a loading control. Confocal analysis of LAMP2 staining (red) in GSCs #1, #4, #12 treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Alternatively, GSC#9 were either treated for 16 hours with H2O or Z-VRPR-FMK (75 µM). Additionally, cells were transfected with non-silecing duplexes (sic) or MALT1 and BCL10 targeting siRNA duplexes (siMALT1 and siBCL10). Alternatively, lysotracker staining (red) was used to track for lysosomes in either GSC#9 expressing either wild-type (WT) or C464A FLAG-MALT1 (green). Scale bar: 10 μm. Quantification of LAMP2 staining pixel intensity on GSC#9 treated as described in panel (D). Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>30. Cryosections from GSC#9-xenografted tumors in vehicle and MPZ-challenged animals (as described in Fig 2J) and assessed for LAMP2 staining (green). Nuclei (DAPI) are shown in blue. Scale bar: 10 μm. Western-blot analysis of LAMP2 was performed in tumor lysates. GAPDH served as a loading control. Confocal analysis of lysotracker staining (red) in GSC#9 treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Alternatively, GSC#9 were either treated for 16 hours with H2O or Z-VRPR-FMK (75 µM) (upper panel) or transfected with sic and siMALT1 (bottom panel). As indicated, number of lysotracker-positive puncta and lysotracker pixel intensity (arbitrary unit, AU) were quantified per cell. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>30. Nuclei (DAPI) are shown in blue. Scale bars: 10 μm.
Redistribution of γ-TuRC-binding factors to the GA in the absence of the centrosome A Control (top) and centrinone-treated (bottom) RPE-1 cells double stained for either AKAP450, CDK5Rap2, PCNT or CEP192 (all shown in green) and giantin (red) as a Golgi marker.Figure 5Control (top) and centrinone-treated (bottom) RPE-1 cells double stained for either AKAP450, CDK5Rap2, PCNT or CEP192 (all shown in green) and giantin (red) as a Golgi marker. DNA was counterstained with DAPI and is shown in blue. Single channel (left, green) and merged images (right) are shown in each case. White arrows indicate the centrosome (CTR). IF images of control and centrinone-treated RPE-1 cells treated with NZ to induce GA fragmentation and double stained with either anti-AKAP450, anti-CDK5Rap2 or anti-PCNT (all shown in green) and anti-giantin antibodies (white). Enlarged views of the boxed areas are presented at the bottom with or without the signal of the Golgi marker. The yellow dashed line indicates the contour of the nucleus. Arrowheads point to accumulation of the respective proteins in Golgi membrane surfaces. Box-and-whisker plots showing quantification of association between the indicated protein and Golgi elements Top and bottom ends of the boxes represent 75th and 25th percentiles, and whiskers represent 90th and 10th percentiles. The median is depicted with a solid line. Individual Golgi elements from at least 14 cells were delineated (>500 elements/experiment) and the intensity of each protein spot associated with each Golgi element was measured. Data were collected from two independent experiments and normalized to WT mean. MT-regrowth experiment after NZ treatment in centrinone-treated cells. High magnification images at the bottom are representative of a triple staining with α-tubulin (green), GM130 (blue) and the indicated protein (red) and show MTs growing from the surface of the GA. Double labelling without α-tubulin are also shown (top) to allow better visualization of PCM-protein spots on Golgi elements acting as MT nucleation sites (arrowheads). Centrinone-treated pcnt KO, c5rap2 KO and akap KO cells, were incubated with NZ for 3h, fixed and double labelled for GM130 and either CDK5Rap2 or PCNT as indicated (n=3). MT regrowth assay in centrinone-treated WT, pcnt KO and akap KO RPE-1 cells stained with antibodies to EB1 and giantin. At right, quantification of EB1 intensity at the Golgi membranes in WT and pcnt KO cells treated with centrinone, as a measure of MT-nucleation from the GA. Data were collected from two independent experiments and normalized to WT mean.
Figure 1(A) Quantitative analysis of the Aβ1-40 protein in the TBS (*p=0.0212), Triton-X (p=0.0544) and SDS (**p=0.0063) fractions of brain homogenates from male (n=7) and female (n=10) APP23 mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test. (B) Stereological analysis of cortical area covered by 4G8-positive plaques in male (n=10) and female (n=8) APP23 mice (left) and representative images (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, ***p=0.0004. (C) Fluorescence intensity based analysis of pFTAA-stained Aβ plaques in the cortex of male (n=10) and female (n=8) APP23 mice (left) and representative images (right), scale bar = 1 mm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, **p=0.0011. (D) Stereological analysis of cortical area covered by Congo Red-positive plaques in male (n=10) and female (n=8) APP23 mice (left) and representative images (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, ***p=0.0001. (E) Native filter assay analysis of TBS (p=0.2453), Triton-X (p=0.0604), SDS (*p=0.0196) and formic acid (FA) (**p=0.0057) fractions from male (n=8) and female (n=7) APP23 mouse brain homogenates. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test between the same fractions. Data points were taken from graphs analysing APP23p40+/+ against APP23p40-/- mice.
Survival, gain of weight and mobility plots for Armadillidium vulgare during 75 days post-injection.Figure 1Survival, gain of weight and mobility plots for Armadillidium vulgare during 75 days post-injection.There were no significant differences between animals injected with wVulC, wDil or control treatment for all these life history traits: Survival (A), Gain of weight (B) and Mobility (C).
Figure 5A-C The kinetic block of SV endocytosis at excitatory synapses depleted of PI(3) P depends on prior neuronal activity and Ca2+. VPS34IN1-induced defects in SV endocytosis are reverted by parallel treatment with TTX (0.2 µM) in A and EGTA (2 mM) in B and AP5 (20 µM) in C, in Synaptophysin-pHluorin-expressing neurons N = 3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM ; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 D Overexpression of dominant-negative Rab5 (Rab5DN), but not constitutively active Rab5 (Rab5CA), rescues the progressive accumulation of Synaptophysin-pHluorin on the surface of VPS34IN1-treated hippocampal neurons stimulated and analyzed as in A-C. N = 3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 E Levels of various synaptic proteins in DMSO (0.1%) and VPS34IN1 (10 µM) treated cultured cerebellar granule neurons analyzed by western blot. VPS34 inhibition results in p35 processing to hyperactive p25, a reaction blocked by Calpain inhibitor ALLN (100 µM). F-H Calpain inhibition by ALLN (100 µM) in F or Calpain 2 knockdown via lentiviral shRNA (shCAPN2) in H rescue the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface induced by VPS34IN1. Calpain 1 knockdown via lentiviral shRNA (shCAPN1) in G had no impact on VPS34IN1-induced kinetic delay of SV endocytosis. N = 3 independent experiments (≥ 11 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM ; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Immunoblotting for procaspase-1 and activated caspase-1 (p10), pro-IL-1β and cleaved IL-1β (p17) in supernatants (Sup) and cell lysates (Lys) from resting or LPS-primed BMMs generated from WT, Nlrp3−/−, Pycard−/− (a) or Nlrc4−/− (b) mice stimulated with PA-BSA (0.figf2Immunoblotting for procaspase-1 and activated caspase-1 (p10), pro-IL-1β and cleaved IL-1β (p17) in supernatants (Sup) and cell lysates (Lys) from resting or LPS-primed BMMs generated from WT, Nlrp3−/−, Pycard−/− (a) or Nlrc4−/− (b) mice stimulated with PA-BSA (0.2 or 0.5 mM) as indicated at top. Data are representative of two independent experiments.
Figure 4(A) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3). Cytoplasmic and nuclear extracts were isolated at the indicated time points post infection, and levels of ICP27, RCC1, and GAPDH were determined by Western blotting. (B) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 10). The cells were fixed at the indicated time points post infection, and stained with DAPI and an antibody against ICP27, and visualized by confocal microscopy. Scale bar 10 μm. (C, D) THP1 cells were infected with HSV-1 KOS, ΔICP27 or HSV-1 ΔNLS. The localization of the major NLS is illustrated in panel C. Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. (E, F) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3). Cytoplasmic (E) and nuclear extracts (F) were isolated at the indicated time points post infection. Western blotting was used to determine levels of phospho- and total TBK1 were determined in the cytoplasmic fractions and levels of phospho-IRF3 and RCC1 in the nuclear fractions. (G-I) HEK293T cells were (G) transfected with STING, TBK1, IRF3-5D, and ICP27, together with the reporter gene constructs indicated. Reporter gene activity was measured 24 h post transfection. (G-I) HEK293T cells were ((H) transfected with STING and stimulated for 16 h with cGAMP (4 μg/ml) together with the reporter gene constructs indicated. Reporter gene activity was measured 24 h post transfection. (G-I) HEK293T cells were (I) transfected with TRIF, MAVS, STING, and ICP27, together with the reporter gene constructs indicated. Reporter gene activity was measured 24 h post transfection.
Figure 4(A) After serum-starvation for 24 h, A549 and H1299 cells overexpressing QKI-5 were treated with or without TGF-β1 (5 or 10 ng/ml ) for 24 h. Then, EMT markers N-cadherin and Snail expression were analyzed using western blot. β-actin served as an internal control. (B) QKI-5-overexpressing A549 and H1299 cells and control cells were treated as above and incubated with a mouse anti-F-actin antibody, and then stained with a FITC-conjugated anti-mouse IgG (green, for F-actin). Cell nuclei were counterstained and visualized with DAPI (blue). Scale bar, 100 μm. (C-F) Wound-healing migration assays were performed on QKI-5-overexpressing A549 and H1299 cells as well as control cells The wound healing was recorded (C and E) and quantitatively measured (D and F) at least six times. (G and H) In the presence or absence of TGF-β1, QKI-5-overexpressing A549 and H1299 cells were allowed to migrate through a polycarbonate membrane in Transwells. After 24 h, migrated cells were stained, photographed and counted in at least four random fields under a light microscope. Representative images (left) and the migrated cell numbers (right) were presented. (I and J) QKI-5-overexpressing A549 and H1299 cells were treated and allowed to invade through Matrigel-coated membrane in Transwells. Invasive cells were determined
Dual‑AAV mediated otoferlin gene delivery partially restores auditory function in deaf Otof-/- mice.Figure 3Representative ABR wave traces in response to broadband click sound stimuli from otoferlin dual‑AAV‑TS and dual‑AAV‑Hyb injected CD1B6F1 Otof-/- animals. AAV2/6.eGFP (+AAV.eGFP) and dual‑AAV‑TS injected CD1B6F1 wild‑type, and non‑injected control Otof-/- littermate (-AAV) mice served as controls. SP: summating potential; ABR waves are indicated from I-V Average ABRs evoked by 80 dB SPL click sound stimuli for 20 clicks/s ABR click sound (C thresholds in otoferlin dual‑AAV treated Otof-/- mice compared to wild‑type and non‑treated Otof-/- control animals ABR tone burst (D) thresholds in otoferlin dual‑AAV treated Otof-/- mice compared to wild‑type and non‑treated Otof-/- control animals. In D, the two best animals are depicted with open circles. Animals with thresholds exceeding the maximum loudspeaker output (arrows) of 100 dB SPL for clicks and 90 dB SPL for tone bursts were set to 110 dB SPL and 100 dB SPL, respectively. Apical and basal cochlear turns are indicated as Apex and Base, respectively Summed ABR wave I-V amplitudes at different click sound intensities in otoferlin dual‑AAV injected, non‑injected Otof-/-, and wild‑type control mice Summed ABR wave I‑V amplitudes of individual dual‑AAV‑TS treated CD1B6F1 Otof-/- animals (n=8 animals plotted against their full-length otoferlin IHC transduction rate
a, Representative confocal images of endogenous OFD1 puncta with cilium marker acetylated tubulin (Ac-tubulin), or centriole marker &ggr-tubulin in hTERT-RPE1 cells in normal medium (Un) or subjected to 24 h serum starvation (SS).f2a, Representative confocal images of endogenous OFD1 puncta with cilium marker acetylated tubulin (Ac-tubulin), or centriole marker γ-tubulin in hTERT-RPE1 cells in normal medium (Un) or subjected to 24 hserum starvation (SS). b, Representative confocal images of endogenous OFD1 and acetylated tubulin in Atg5+/+ and Atg5−/− MEFs subjected to 24 hserum starvation. a, b, Data shown represent mean ± s.d. percentage of cells with centriolar satelliteOFD1 for 100 cells per well in triplicate samples. ***P 0.001, two-tailed unpaired student's t-test. Similar results were observed in three independent experiments.
Figure 1(A) Schematic of the α11.2 subunit of Cav1.2 (top) and the α11.2-derived GST fusion proteins covering the C-terminus (bottom). (B) Pull-Down of GST-tagged α11.2 segments (top immunoblot; IB) by immobilized MBP-β2AR C-terminus (residues 326-413 of human β2AR). GST fusion proteins were detected by an anti-GST antibody and MBP fusion proteins by an anti-MBP antibody. Middle IB shows that comparable amounts of the various GST fusion proteins had been added to the resin samples and bottom IB illustrates equal loading of all amylose resin samples with MBP-β2AR.(C) Quantification of (B) (***p<0.001, ANOVA). (D) Pull-Down of GST-CTC but not GST-CTD or GST-CTE (middle IB) by immobilized MBP-β2AR C-terminus (left) but not MBP alone (right), all of which were present at comparable amounts (bottom and top IBs, respectively). GST fusion proteins were detected by an anti-GST antibody and MBP fusion proteins by an anti-MBP antibody.(E) Quantification of (D) (***p<0.001, ANOVA). (F) IP of β2AR in the presence of 10 μM GST (Control; Ctr) or GST-tagged C-terminal fragments as indicated. CT4 and CTC specifically displaced α11.2 (top of IB) but not GluA1 (middle of same IB) from β2AR (bottom of same IB). Use of non-specific IgG (left lane in right panels) indicates specificity of IP.(G, H). For quantification of coIPs, α11.2 and GluA1 immunosignals were normalized to β2AR signals (***p<0.001, ANOVA).(I) Representative IB showing amounts of the GST fusion proteins that were added in (F), as detected by anti-GST antibodies.
Implication of the amino-terminal region in aggregation(A) The level of aggregation relative to piliation level is represented as the Log2 of the ratio between the frequency of each single point mutation in the aggregation library relative to the frequency of the same mutation in the piliation library.Figure 2The level of aggregation relative to piliation level is represented as the Log2 of the ratio between the frequency of each single point mutation in the aggregation library relative to the frequency of the same mutation in the piliation library. Each round dot represents a point mutant. Mutation type is color-coded as indicated in the legend. Squares on the x-axis correspond to the original amino acids in the SB sequence with the protein secondary structure indicated below. Dotted lines represent a two-fold threshold value. Scanning electron microscopy of single bacteria on a cellulose filter. Scale bars: 500 nm. Two representative immunofluorescence labelling of TFP. Scale bar: 1 µm. Piliation per bacterium as a function of the proportion of piliated bacteria normalized to pilESB values as measured by flow cytometry using the 20D9 monoclonal antibody. Black dots indicate all the individual de novo generated PilE mutants that were characterized. Blue dots indicate mutants of the piliation machinery and green dots mutants in the α1N region. Solid lines indicate 95% confidence interval. Quantification of aggregation normalized to pilESB for a subset of outliers and corresponding pilT double mutants. Mean values ± SEM are indicated for each strain. N≥3 independent experiments. Paired t-test. p<0.05 (*), p<0.01 (**).
Figure 1(A) Structure of full-length, nucleotide-free, farnesylated hGBP1 (PDB entry 6k1z) and GDP·AlFX-bound LG-domain dimer (PDB entry 2B92). Insert (a) shows the farnesyl moiety and the triple arginine stretch (3R = R584-586). Insert (b) highlights residues required for nucleotide binding and hydrolysis. (B) Experimental design: fluorescently labeled recombinant hGBP1 variants and nucleotides were mixed with broth-cultured live or fixed bacteria; bacterial binding was monitored by confocal microscopy. (C) Confocal images of formaldehyde fixed GFP+ S. flexneri following 20 min incubation with 2 mM GTP and 10 µM Alexa-Fluor647-labeled protein. Bacteria associated with hGBP1 mutants after 20 min were quantified. Mean frequencies ± SEM of combined data from two independent experiments are shown. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ***, P ≤ 0.001. Scale bars equal 5 µm. Flow diagram depicts effects of K51A, R48A, and H74A hGBP1 mutations on nucleotide binding and hydrolysis.
Figure 6Schematic of Lmnb1-KO in NPCs. After the introduction of LV, NPCs were kept in proliferative conditions with FGF2 and EGF. MA plot of differentially expressed genes between control and Lmnb1-KO NPCs. C, D. Gene ontology analyses in LaminB1-regulated genes. E. Fraction of direction of gene regulation by LaminB1. Genes were allocated dependent on where LaminB1 interacts with genes. Red bars indicated upregulated genes and blue bars indicate downregulated genes after lamin B1 knockout. F. Examples of lamin B1-directed genes in NPCs. Blue bars indicate laminB1-LAD.
Fig.Figure 4(A) Schematic diagram of lentiviral uSTAT5 knock-down in HPC7 cells. (B) Depletion of uSTAT5 was accompanied by the appearance of large cells that stain positively for Acetylcholinesterase (AchE). Scrambled shRNA (scr), shSTAT5_5 (sh_5) or shSTAT5_7 (sh_7). (C) Increased percentage of polyploid cells following uSTAT5 depletion. Bars represent means ± SD from three independent experiments. (D) Increased percentage of cells expressing high levels of CD41 and CD61 following uSTAT5 depletion. Bars represent means ± SD from three independent experiments. (E) Strategy for assessing the effect of increasing uSTAT5 on megakaryocytic differentiation of HPC7 cells. (F) Western blot analysis of HPC7 cells expressing empty vector (EV), wild type STAT5B (WT) or mutant STAT5B (Y699F). EV and Y699F expressing cells exhibit similar levels of STAT5 phosphorylation following TPO treatment (100ng/ml for 30min). (G) Expression of the Y699F mutant repressed the appearance of large cells that stained for acetylcholinesterase (AchE). Scale bars represent 25μm. (H) The Y699F mutant increased the percentage of polyploidy cells. Bars represent means ± SDs from three independent experiments. (I) The Y699F mutant increased the percentage of cells expressing high levels of CD41 and CD61. Bars represent means ± SD from three independent experiments. (J) Quantitative RT-PCR showing that the Y699F mutant did not alter induction of pSTAT5 target genes by endogenous pSTAT5 following TPO stimulation (100ng/ml for 30 min). Bars represent means ± SDs from three independent experiments. (K) Quantitative RT-PCR showing that the Y699F mutant repressed induction of uSTAT5 target genes following TPO treatment. Bar graphs represents means ± SDs from three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 2A. Representative spinning disk confocal live-cell imaging time series images of U2OS PA-GFP-α-tubulin cells conditionally co-expressing KIF4A shRNA and RNAi-resistant mCherry-Kif4A variants induced using doxycycline. Chromosomes were stained using SiR-DNA. White arrowheads highlight poleward flux of the photoactivated regions. Scale bar, 10 µm. Time, min:sec. B. Representative immunoblot of cell lysates obtained before and after doxycycline induction to validate the efficiency of KIF4A shRNA construct and expression of the RNAi-resistant mCherry-KIF4A variants. The anti-KIF4A antibody was used, together with anti-vinculin antibody as a loading control. C. Quantification of MT-flux upon shRNA-mediated depletion of KIF4A alone or in combination with conditional expression of the RNAi-resistant mCherry-KIF4A variants. The bars in graph represent mean ± SD. N (number of cells, number of independent experiments): uninduced shKIF4A (48, 4), shKIF4A (60, 4), uninduced KIF4A WT (36, 5), shKIF4A + KIF4A WT (43, 5), uninduced KIF4A K94A (42, 7), shKIF4A + KIF4A K94A (38, 6), uninduced KIF4A ΔZip1 (32, 4), shKIF4A + ΔZip1 (30, 4). D. Model illustrating chromosome arms-localized KIF4A driving MT-flux.
Functional analysis of precisely defined agonist‐liganded ARBEsSchematic representation of ARBE wild‐type (WT) and deletion mutant (Mut) reporters.Figure 3A Schematic representation of ARBE wild‐type (WT) and deletion mutant (Mut) reporters. B ARBE reporter constructs were transiently transfected into LNCaP cells. Cells were stimulated with DHT (100 nM), a synthetic androgen R1881 (10 nM), or vehicle for 16-24 h, and luciferase activities were measured. The results were presented as the mean ± SD of the quadruplicate transfections. The genomic positions of ARBEs are also shown. C UCSC genome browser showing signals on ChIP‐exo defined TMPRSS2 −13.5 kb enhancer ARBE. D Enhancer activity was compared between literature‐based ARE and Exo‐defined ARBE in TMPRSS2 locus by reporter gene assay. E ARBE2 and ARBE4 were validated in TMPRSS2 enhancer through WT or Mut construct transfections. The results were presented as the mean ± SD of the quadruplicate transfections. F Two ARBE1 regions were changed to ARBE2, ARBE3, or ARBE4 through mutations at nucleotide 5 and/or 6. Cells were stimulated with DHT (100 nM), R1881 (10 nM), or vehicle for 16-24 h, and luciferase activities were measured. The results were presented as the mean ± SD of the quadruplicate transfections. The genomic positions of ARBEs are also shown.