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Figure 1. A dual-fluorescence reporter cassette for real-time tracking of adaptive mutations of different types. ; (A) Reporter cassette construct for chromosomal insertion. p0 = 188 bp random DNA sequence, RBS = ribosomal binding site, hairpins = transcriptional terminators, tetA-yfp = selected gene, cfp = constitutively expressed amplification reporter. A, B, C, D = intergenic chromosomal insertion loci, oriC = origin of replication. (B) Immediate chromosomal neighborhoods of loci A-D. Black arrows = essential genes. White arrows = non-essential genes. Grey arrows = no essentiality data available. Patterned arrow (yoeD) = pseudogene. Orange = cryptic prophage CP4-44. Green = origin of replication (oriC). Chromosomal neighborhoods of loci B, C, and D are shown reversed with respect to conventional chromosome coordinates, so that the orientation relative to the reporter cassette is shown in the same way for all four loci. Reporter cassette genes are not drawn to scale. (C) Example fluorescence trajectories of rescued populations with YFP or YFP+CFP (amplification) fluorescence phenotype. RFU = relative fluorescence units (see Methods), yellow and blue lines = YFP and CFP fluorescence, dotted lines = threshold for phenotype classification. (D) Increase of tetracycline concentration in ten-day experiment, normalized to strain-specific minimal inhibitory concentration (MIC, dotted line). (E) qPCR validation of CFP fluorescence as an indicator of extent of amplifications. x-axis: tetA-yfp copy number as determined by qPCR on genomic DNA of rescued population with a YFP+CFP fluorescence phenotype. Error bars = SD of technical qPCR triplicates. r is the Pearson correlation coefficient and P its p-value. RFU = relative fluorescence units, line = linear fit.

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Figure 2. Assay for red blood cell (RBC) cholesterol accessibility. ; (A) Schematic of anthrolysin O (ALO) domains. Domain 4 (ALOD4) binds cholesterol but does not oligomerize or form membrane-lysing pores. In fALOD4, Alexa Fluor 488 dye is covalently attached to an engineered cysteine near the NH2-terminus of domain 4. Purified proteins (5 µg) were subjected to SDS-PAGE (15%) and visualized by Coomassie staining (left) or fluorescence scanning (LI-COR) at 600 nm (right). (B) Flow cytometry analysis of fALOD4 binding to RBCs. RBCs (2.5 × 105 RBCs in 500 μl buffer D) were incubated for 3 hr at 4°C with fALOD4 at the indicated concentrations. Fluorescence was measured by a FACSCalibur flow cytometer as described in the Materials and methods. Forward light scatter (FSC), side light scatter (SSC), and Alexa 488 fluorescence measurements for 10,000 RBCs were acquired on the flow cytometer. Median Alexa 488 fluorescence per cell was calculated using FlowJo software. AU, arbitrary units; RFU, relative fluorescence units. (C) Dose response (left) and time course (right) of fALOD4 binding to RBCs. Each reaction was set up as described above using either the indicated concentrations of fALOD4 (right) or 250 nM fALOD4 (left). After incubation at 4°C for 3 hr (left) or for the indicated times (right), fALOD4 RBC binding was measured by flow cytometry. Hemolysis during fALOD4 binding reactions was determined by measuring the release of hemoglobin as described in the Materials and methods. 100% hemolysis is defined as the amount of hemoglobin released after treatment of RBCs with 1% (w/v) Triton X-100. Data points represent means of three independent measurements of the same sample (technical replicates). Error bars, which are often not visible due to the small variation, represent the SEM. The experiment was repeated three times and the results were similar. (D) Effect of RBC cholesterol modulation on RBC fALOD4 binding. (Left) RBCs were not treated (green circle) or treated with either hydroxypropyl-β-cyclodextrin (HPCD) or cholesterol/methyl-β-cyclodextrin (MCD) to reduce or increase the cholesterol content of RBCs. The fALOD4 binding assay was performed as described in the Materials and methods using 250 nM of fALOD4. Lipids were extracted from ghost membranes isolated from the RBCs, and the molar percentage of cholesterol was measured as described in the Materials and methods. The dashed line indicates the cholesterol content of untreated RBCs. Hemolysis was measured as described in the Materials and methods. Inset: Data for the experiment plotted on a logarithmic-linear scale. (Right) RBCs were treated with HPCD to remove cholesterol and both fALOD4 binding and cholesterol content were measured as described in the Materials and methods. The fALOD4 binding per cell and cholesterol content (mole %) in untreated RBCs was set to 100%. Data points represent the mean of three independent measurements of fALOD4 binding and a single measurement of RBC cholesterol content. Error bars represent the SEM. RFU, relative fluorescence units. The experiments were repeated three times and the results were similar.

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Figure 5. Ectopic AURKA increases mitochondrial respiration together with the abundance and the functionality of the mitochondrial respiratory chain. ; (A) (Top) Western blot of total lysates of HEK293 cells transfected as indicated and (bottom) corresponding quantification of the relative abundances of the indicated oxidative phosphorylation complexes subunits, representing the abundance of the five oxidative phosphorylation complexes. n = 3 independent experiments. (B) Mitochondrial respiration of HEK293 cells over-expressing GFP or AURKA-GFP. n = 3 independent experiments. (C) Percentage of MCF7 cells over-expressing or silenced for AURKA and with activated MAP1LC3A-II and analysed by flow cytometry. n = 3 independent experiments. (D) Proteasomal activity in MCF7 cells transfected as indicated and analysed by flow cytometry. n = 3 independent experiments. RFU: relative fluorescence units. (E) MitoTimer red/green ratio in MCF7 cells transfected as indicated. n = 3 independent experiments. (F) Red fluorescence of the mitochondrial potentiometric probe JC-1 in MCF7 cells transfected as indicated and analysed by flow cytometry. The decrease in red JC-1 fluorescence provides a readout of ΔΨloss. n = 3 independent experiments. (G) TMRM relative fluorescence of MCF7 cells transfected as indicated and treated with DMSO (basal conditions) or carbonyl cyanide m-chlorophenyl hydrazone (CCCP). n = 3 independent experiments. (H) Mitochondrial respiration of HEK293 cells over-expressing a control or an AURKA-specific shRNA. n = 3 independent experiments. (I) Percentage of live, apoptotic and dead MCF7 cells analysed by flow cytometry and identified by the incorporation of Annexin V. n = 3 independent experiments. (L) Mitochondrial respiration of HEK293 cells treated with DMSO or MLN8237 at a concentration of 100 nM for 3 hr or of 250 nM for 10 min. n = 3 independent experiments. (M) Cartoon diagram of AURKA silencing or overproduction acting differentially on key mitochondrial functions. Green arrows: upregulation; red arrows: downregulation. UPS: ubiquitin-proteasome system; CIV: mitochondrial complex IV. Data represent means ±s.e.m. ***p

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Figure 8. BMP inhibition partially rescues the cellular defects of YAPTAZ knockdown HUVECs. ; (A) Luciferase reporter assay for Notch activity in YAP/TAZ knockdown HUVECs and controls treated with 0.1 μM DBZ or DMSO. Data are mean ±SEM. p values were calculated using unpaired t-test. n ≥ 3 biological replicates. (B) Quantification of wound closure at 16 hr for HUVECs knocked down for YAP/TAZ and treated with 0.1 μM DBZ or DMSO. Data are mean ±SD. p values were calculated using unpaired t-test. n = 6 biological replicates. (C) Schematic of the BMP inhibitors used depicting preferential sites of inhibition. Alk1fc, ENGecd and Gremlin preferentially bind extracellular BMPs. K02288 and Ldn193189 are kinase inhibitors. (D) Luciferase reporter assay for BMP activity in YAP/TAZ knockdown HUVECs and controls treated with 25 ng/mL Alk1fc, 0.25 μg/mL ENGecd, 0.1 μg/mL Gremlin, 1 μM K02288, 1 μM Ldn193189 and DMSO. Data are mean ±SEM. p values were calculated using unpaired t-test. n ≥ 3 biological replicates. (E) Quantification of wound closure at 16 hr for HUVECs knocked down for YAP/TAZ and treated with 1 μM Ldn193189 or DMSO. Data are mean ±SD. p values were calculated using unpaired t-test. n = 6–7 biological replicates. (F) Permeability of HUVECs knocked down for YAP/TAZ and treated with 1 μM Ldn193189 or DMSO to 250 kDa fluorescent dextran molecules. Data are mean ±SD. RFU, relative fluorescence units. p values were calculated using unpaired t-test. n = 6 biological replicates. (G, H) HUVECs knocked down for YAP/TAZ and treated with 1 μM Ldn193189 (H) or DMSO (G) stained for VE-Cadherin. (G’, H’) different colours mark different cells. Red arrowheads, fingers. Red arrow, reticular junction. Scale bar G, H, 50 μm. Scale bar G’, H’, 10 μm. (I) Morphological analysis of VE-Cadherin labelled cell junctions in HUVECs knocked down for YAP/TAZ and treated with 1 μM Ldn193189 or DMSO control. Data are mean ±SD. p values were calculated using unpaired t-test. n = 3 biological replicates; n ≥ 45 patches of VE-Cadherin stained HUVECs per condition per replicate.

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Figure 4. Accumulation of MIH in Opsin9 mutant gonads. ; Quantification of MIH loss from gonad ectoderm after light stimulation. All images were summed from 10 confocal Z planes over 4 µm, acquired on the same day using constant settings. Immunofluorescence of fixed, methanol –treated gonads using anti-PRPamide antibodies (MIH; white) was quantified in putative MIH-Opsin cells identified by typical morphology revealed by anti-alpha tubulin (magenta) antibodies. (A) Representative images of wild type Clytia gonad showing reduction in MIH fluorescence after light stimulation. (B) Distribution of RFU (Relative Fluorescence Units) values obtained for each cell analysed in the two conditions (number of cells analysed: n = 226 and n = 282, respectively). (C) Graph showing the medians of the data in (B). Limits correspond to first and third quartiles. The Mann-Whitney U test showed a significant difference between conditions (at p

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Figure 2. C94 in TAPBPR is not involved in an intramolecular disulphide bond ; (A) Amino acid sequences of human TAPBPR (NP_060479.3) and tapasin (AAC20076.1) were aligned using ClustalW. Cysteine residues are marked in yellow boxes, with the cysteine in tapasin that interacts with ERp57 and the predicted unpaired cysteine in TAPBPR highlighted in red. The positions of the cysteine residues in TAPBPR and tapasin are labelled above and below, in blue or green, respectively. (B) Structure of tapasin (Protein Data Bank ID: 3F8U), with the cysteines involved in intramolecular disulphide bonds highlighted in yellow and the free cysteine C95 highlighted in red. (C) FFAS model for TAPBPR (Hermann et al., 2013), with potential disulphide bridges highlighted in yellow and the predicted free cysteine C94 highlighted in red. (D) Lysates from a HeLaM cell panel expressing cysteine-mutant TAPBPR molecules were resolved under non-reducing (no 2-ME) or reducing (+2-ME) conditions, then blotted for TAPBPR (using mouse anti-TAPBPR), MHC class I heavy chain (using HC10), or calnexin as a loading control. The antibody used to detect TAPBPR was raised against the membrane distal domain (aa 23–122); therefore, it is unlikely that the lack of detection of C300 and C361 is due to a lack of antibody recognition of these IgC domain mutants. Data shown in (D) are representative of three independent experiments. (E and F) Size exclusion chromatogram of TAPBPRWT and TAPBPRC94A purified from cell culture supernatant. The protein peaks were analysed by SDS-PAGE followed by Coomassie staining. (G) Differential scanning fluorimetry of TAPBPRWT and TAPBPRC94A demonstrates equivalent thermal denaturation profiles. WT: wild-type; WB: western blot; RFU: relative fluorescence units.

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Figure 4. Distribution of fALOD4 binding to RBCs in individuals with low, medium and high binding. ; (A) Flow cytometry analysis of fALOD4 binding to RBCs from three individuals with low (top), medium (middle) and high (bottom) fALOD4 binding. Forward light scatter, side light scatter, and Alexa 488 fluorescence measurements of 10,000 RBCs were acquired using a FACSCalibur flow cytometer as described in the Materials and methods. Fluorescence data are presented as both a dot plot (middle) and histogram (right). AU, arbitrary units; RFU, relative fluorescence units. (B) Relationship between RBC number and fALOD4 binding in three individuals with low (green), medium (blue) and high (orange) binding. Samples were collected on the same day and the fALOD4 binding assay was performed as described in the legend to Figure 1. Error bars represent the mean ± SEM of three measurements from each blood sample. The experiment was repeated once and the results were similar. (C) RBC cholesterol content and fALOD4 binding to RBCs. RBC ghost membranes were prepared from RBCs of 73 healthy, unrelated individuals. Total lipids were extracted from the membranes and the molar percentage of cholesterol was measured as described in the Materials and methods. fALOD4 binding values were normalized to the binding values obtained from the reference blood sample. Shown is the Spearman correlation between fALOD4 binding, normalized to the reference blood sample, and RBC membrane total cholesterol expressed as mole % of total lipids. Data points represent the means of three independent measurements of fALOD4 binding and a single measurement of RBC cholesterol content. The experiment was repeated once and the results were similar. nRFU, normalized relative fluorescence units.

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Figure 3. Phosphorylation of ubiquitin-specific protease-14 (USP14) by Akt activates USP14 DUB activity. ; (A) Akt activates USP14 DUB activity in vitro. USP14 protein (1 μg) was incubated with or without active Akt (1 μg) in kinase assay buffer in a total volume of 50 μL for 1 hr at 30°C, then the reaction mixtures were subjected to Ub-AMC assay. RFU, relative fluorescence units. (B, C) Akt activates USP14 in cells. USP14 was immunoprecipitated from HEK293T cells coexpressed with activated Akt (B) or treated with 10 μM MK2206 for 4 hr (C) and then eluted with HA-peptide following Ub-AMC hydrolysis assay. (D) Activation of USP14 by stimulating cells with IGF-1. HEK293T cells were serum-starved and pretreated with or without Akt inhibitor MK2206 (1 μM) for 30 min before stimulation with IGF-1 (100 ng/mL) for 30 min. USP14 was then immunoprecipitated and eluted with HA-peptide. The activity of USP14 was determined using Ub-AMC hydrolysis assay. (E) USP14 activation by Akt is blocked by S432A mutation. Ub-AMC hydrolysis assay of wildde type USP14 or S432A mutant in the presence or absence of active Akt. (F) Ub-AMC hydrolysis assay of bacterially expressed and purified wild type USP14 or S432E mutant. (G) Lineweaver–Burk analysis of USP14 S432E, obtained by measuring the initial rates at varying Ub-AMC concentrations (see Figure 3—figure supplement 2E for reference). (H) The activity of phospho-mimetic USP14 mutant can be further stimulated by the presence of proteasome. Ub-AMC hydrolysis assay of wild type USP14 or S432E mutant in the presence or absence of Ub-VS-treated human proteasome (VS-proteasome (see Lee et al., 2010); 1 nM). Ptsm, 26S proteasome.

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Figure 4. SBT5.2(a), but not SBT5.2(b), shows serine protease activity. ; (a) Western blot analysis shows expression of HA-tagged SBT5.2(a), SBT5.2(b) and their catalytic mutant versions in N. benthamiana, as indicated. Ponceau S staining confirms equal loading. Molecular mass markers in kilodaltons are indicated on the right. (b) Fluorimetric assay to detect protease activity following incubation of Arabidopsis protoplasts expressing the indicated proteins with fluorescein isothiocyanate (FITC)-conjugated casein (top). RFU: relative fluorescence units. Error bars indicate SEM. Lowercase letters indicate significant differences as determined by Bonferroni-corrected p-values (p

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A novel lectin from Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine

• Authors : Shuai Jiang; Yalin Yin; Yijie Chen

Subjects: RFU, relative fluorescence units; fungal lectin; Biochemistry

• Source: Biochemical Journal

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