Journal: Nature Communications

Article Title: The Flemmingsome reveals an ESCRT-to-membrane coupling via ALIX/syntenin/syndecan-4 required for completion of cytokinesis

doi: 10.1038/s41467-020-15205-z

Figure Lengend Snippet: Proteomics of highly pure and intact post-abscission midbodies revealed known and previously unknown proteins enriched in this organelle. a Midbody remnant purification. HeLa cells (upper left picture) expressing GFP-MKLP2, a kinesin enriched in midbodies (MB) and midbody remnants (MBRs) were EDTA-treated (Total). After 70 g centrifugation, the supernatant (SN) containing MBRs was processed either (1) by differential centrifugations leading to MBR-enriched fraction (MBRE) or (2) subjected to flow cytometry sorting to purify GFP-positive MBRs (MBR+) and their GFP-negative counterpart (MBR−). b Representative pseudo-colored profile of flow cytometry sorting of MBRs. The MBR+ (14% total) and SSC-matched MBR− (44% total) were separated from remaining cells (1%). See Supplementary Fig. 1b . c Western blots of same amounts of protein extracts from Total (Tot), MBR-enriched (MBRE), flow cytometry-sorted MBR− and MBR+ populations. Membranes were blotted repeatedly with indicated antibodies. See also Supplementary Figs. 1c and 6 . d Upper left panel: MBR+ population analyzed with cell mask membrane marker. Each individual midbody is positive for GFP-MKLP2 (green) and cell mask (red) Scale bar: 6 μm. Upper right panel: scanning electron microscopy of an isolated MBR. Note the intact and sealed membrane. Lower panels: immunofluorescence stainings of MBR+ for endogenous proteins or membrane marker (red), as indicated. Scale bars: 2 μm. e The Enriched Flemmingsome . Upper panel: merged volcano plot of the mass spectrometry analysis showing the maximum log2(fold change) in x -axis measured between MBR+ and the other fractions (MBRE, MBR−, or Total) and the corresponding –log10(merged p value) in y -axis. color code: proteins significantly enriched in MBR+ when compared with 3 (red), 2 (blue), or 1 (green) of the other fractions. Bottom panel: proteins quantitatively present in MBR+ but not detected in at least two of the other fractions. ALIX (PDCD6IP), syntenin (SDCBP) and syndecan-4 (SDC4) circled in red. f STRING functional association network for the Enriched Flemmingsome . See Supplementary Fig. 3 for details. g GO-term over-representation clusters in the Total Flemmingsome . The size of each bar ( x -axis) corresponds to the number of proteins in each cluster and the red gradient the enrichment p values coming from hypergeometric tests. Gray: p value > 0.1.

Article Snippet: Sample preparation for mass spectrometry HeLa GFP-MKLP2 were detached from flasks with 0.05% trypsin diluted in 0.02% EDTA (25300; Gibco, Invitrogen Life Technologies) and plated at 8 × 105 cells/well on 10-cm dishes for 3 days.

Techniques: Purification, Expressing, Centrifugation, Flow Cytometry, Western Blot, Marker, Electron Microscopy, Isolation, Immunofluorescence, Mass Spectrometry, Functional Assay

Journal: Nature Communications

Article Title: Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing

doi: 10.1038/s41467-018-04525-w

Figure Lengend Snippet: Laminin HBD peptides promote cell adhesion in vitro. a , b A total of 3000 cells/well human lung fibroblasts were cultured a without or b with 5 mM EDTA in FGM-2 culture media containing 1% FBS. c , d A total of 3000 cells/well HUVEC were cultured c without or d with 5 mM EDTA in EBM-2 culture media containing 100 ng/mL VEGF-A165 and 1% FBS. Cells were plated on 1 μg/mL laminin peptide pre-coated non-tissue culture-treated plates and incubated for 30 min at 37 °C. After plate washes, cell numbers were quantified using a CyQUANT assay ( n = 10, mean ± SEM). The signals obtained from BSA-coated wells are normalized to 1, and relative fold increases of cell numbers were calculated. Statistical analyses were performed using ANOVA with Tukey’s test. Kruskal–Wallis test followed by Dunn’s multiple comparison was used in b , c . * p

Article Snippet: Cell adhesion assays were performed using human lung fibroblasts (Lonza) in FGM-2 medium (Lonza) or human umbilical vein endothelial cells (HUVEC; Lonza) in EGM-2 medium (Lonza) supplemented with 1% fetal bovine serum (FBS) and 100 µg/mL VEGF-A165, with or without 5 mM EDTA (Sigma-Aldrich).

Techniques: In Vitro, Cell Culture, Incubation, CyQUANT Assay

Journal: PLoS ONE

Article Title: A Ribosomal Misincorporation of Lys for Arg in Human Triosephosphate Isomerase Expressed in Escherichia coli Gives Rise to Two Protein Populations

doi: 10.1371/journal.pone.0021035

Figure Lengend Snippet: Effect of urea on the intrinsic fluorescence spectra of HsTIM (A and B). Isolation of the two proteins components of HsTIM by size exclusion chromatography (C). In A, the fluorescence spectrum of HsTIM, prepared as described under Material and Methods , was incubated at a concentration of 40 µg/ml in 100 mM triethanolamine, 10 mM EDTA, and 1 mM dithiothreitol (pH 7.4) for 60 min at 25°C with, and without, 6 M urea. At that time the spectra were recorded at an excitation wavelength of 280 nm. B shows the effect of different concentrations of urea on the λmax of its intrinsic fluorescence. C shows the size exclusion chromatography profile (see Methods section) of HsTIM incubated and eluted in a buffer containing 3 M urea. Note that two clearly distinguishable protein peaks were detected; the protein that eluted first was termed P1 and the second P2.

Article Snippet: Generally 300 µl of 20 mM triethanolamine, 0.2 mM EDTA, 200 mM NaCl and 1 mM dithiothreitol (pH 7.4) that contained between 25 to 500 µg of protein were applied and eluted with the same buffer. the columns were calibrated with a gel filtration standard (Bio-Rad) containing the following globular protein markers (molecular mass and retention volumes are reported): thyroglobulin (bovine) (669 kDa, 9.8 ml), γ-globulin (bovine) (158 kDa, 12.9 ml), ovalbumin (chicken) (43 kDa, 15.8 ml), myoglobin (horse) (17 kDa, 17.7 ml), and vitamin B12 (1.35 kDa, 21.1 ml).

Techniques: Fluorescence, Isolation, Size-exclusion Chromatography, Incubation, Concentration Assay

Journal: Scientific Reports

Article Title: Lectin-mediated protocell crosslinking to mimic cell-cell junctions and adhesion

doi: 10.1038/s41598-018-20230-6

Figure Lengend Snippet: Effect of ligand competition with PNPG, and LecA inactivation with EDTA on lectin binding and crosslinking. Time-series of ligand competition with 4-nitrophenyl-α-D-galactopyranoside (PNPG), or lectin inactivation with ethylenediaminetetraacetic acid (EDTA). Liposomes were composed of DOPC:cholesterol:DHPE-TxRed (red) (64.5:30:0.5 mol%, respectively) and 5 mol% Gb3. The TxRed channel (membrane dye) is depicted in the merge, but not as a separate image, as it does not provide additional information. ( a ) Before treatment, vesicles were crosslinked with 100 nM LecA-AF488 (green) for 120 min. ( b ) PBS mock control to exclude dilution effects. ( c ) Top panel: Addition of 1 mM PNPG remained ineffective also after 2 h of ligand competition. Bottom panel: Addition of 10 mM PNPG resulted in the loss of lectin binding outside of contact areas within the first 15 min. Yet, protocellular junctions remained without significant change even after 120 min. ( d ) Addition of 1 mM EDTA was ineffective for the first 15 min but reversed the formation of protocellular junctions within 120 min. Scale bars = 10 µm.

Article Snippet: FSL-Lea (tri) referred to as DOPE-Lea (tri), FSL-biotin referred to as DOPE-biotin, 4-nitrophenyl-α-D-galactopyranoside (PNPG), and ethylenediaminetetraacetic acid (EDTA) were obtained from Sigma-Aldrich.

Techniques: Binding Assay

Journal: Journal of Virology

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (?V?5, ?V?3, and ?3?1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection ▿

doi: 10.1128/JVI.01146-08

Figure Lengend Snippet: Inhibition of KSHV infection by anti-integrin antibodies and soluble integrins. (A) Inhibition of GFP-KSHV infection by anti-integrin antibodies. Monolayers of HMVEC-d and HFF were incubated with 10 μg/ml of MAbs against integrins or integrin subunits for 1 h at 4°C, washed, and infected with GFP-KSHV at an MOI of 5 DNA copies/cell. After incubation for 2 h at 37°C, the cells were washed and incubated for 72 h, and the number of GFP-positive cells was estimated under a fluorescence microscope. Each experiment was done in duplicate, and each bar represents the average plus the standard deviation (SD) of three experiments. (B) Effects of anti-integrin antibodies on KSHV binding. HFF were incubated with anti-integrin antibodies (10 μg/ml) at 4°C for 90 min and incubated with a predetermined concentration of [ 3 H]thymidine-labeled purified KSHV. The labeled KSHV was also mixed with 10 μg/ml of heparin or soluble integrins for 90 min at 4°C and then added to the cells. After incubation for 90 min at 4°C with the virus, the cells were washed, lysed, precipitated with trichloroacetic acid, and counted. The bound virus cpm in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average plus the SD of three independent experiments. The results with HFF are shown. (C) Inhibition of KSHV infection by soluble integrins. KSHV (5 DNA copies/cell) was mixed with 10 μg/ml of heparin or 10 μg/ml soluble integrins for 2 h at 37°C and added to the cells. After 2 h, the cells were washed to remove the unbound virus, treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized viruses, and washed, and total DNA was isolated. The number of KSHV ORF73 copies was estimated by real-time DNA PCR. The cycle threshold values were used to plot the standard graph and to calculate the relative copy numbers of viral DNA in the samples. The data are presented as percentages of inhibition of KSHV DNA internalization obtained when the cells were incubated with the virus alone. Each reaction was done in duplicate, and each bar represents the average ± SD of three experiments. (D) KSHV was mixed with 10 μg/ml soluble integrin combinations for 2 h at 37°C and added to HFF. After 2 h, the cells were washed to remove the unbound virus and treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized viruses, and the internalized viral DNA was estimated by real-time DNA PCR. Each reaction was done in duplicate, and each bar represents the average ± the SD of three experiments.

Article Snippet: Briefly, cells infected with KSHV were collected after 2 h, washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. DNA was isolated from the nuclei using a DNeasy kit (Qiagen) as described previously ( ).

Techniques: Inhibition, Infection, Incubation, Fluorescence, Microscopy, Standard Deviation, Binding Assay, Concentration Assay, Labeling, Purification, Isolation, Polymerase Chain Reaction

Journal: Journal of Virology

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (?V?5, ?V?3, and ?3?1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection ▿

doi: 10.1128/JVI.01146-08

Figure Lengend Snippet: ( A) Effects of anti-CD98 and anti-xCT peptide antibodies on KSHV binding. HMVEC-d, untreated or treated with 10 μg/ml of various antibodies at 4°C for 1 h, were incubated with a fixed concentration of [ 3 H]thymidine-labeled virus in RPMI 1640 for 1 h at 4°C with gentle rotation. For control, [ 3 H]thymidine-labeled KSHV was preincubated with 100 μg of heparin/ml for 1 h at 37°C before addition to the cells. After incubation at 4°C, the cells were washed, lysed, and precipitated with trichloroacetic acid, and cell-associated virus radioactivity (in cpm) was measured. The cpm of bound virus in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average ± standard deviation (SD) of three independent experiments. (B and C) Effects of anti-CD98 and anti-xCT peptide antibodies on KSHV DNA internalization. HMVEC-d were untreated or treated with various concentrations of anti-CD98 MAb (CD98 MAb) and polyclonal antibody (CD98 PAb) or anti-CD71 antibodies (B) or with anti-xCT peptide antibodies (C) at 4°C for 1 h and then infected with KSHV at an MOI of 10 for 2 h. Unbound virus was removed by washing the cells, and the cells were treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized virus. The cells were washed, DNA was extracted, and the internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR. Copy numbers were calculated from the standard graph generated by real-time DNA PCR using known concentrations of a cloned ORF73 gene. Each reaction was done in duplicate, and each bar represents the average ± SD of three experiments. (D) Effect of CD98 ligand, galectin 3, on KSHV DNA internalization. HMVEC-d were first incubated with the CD98 ligand galectin at 5- and 10-μg/ml concentrations for 1 h at 4°C. The cells were washed and then infected with KSHV at an MOI of 10 for 2 h. Internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR. Each reaction was done in duplicate, and each bar represents the average ± the SD of three experiments. KSHV DNA internalization in the absence of any treatment was considered to be 100%.

Article Snippet: Briefly, cells infected with KSHV were collected after 2 h, washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. DNA was isolated from the nuclei using a DNeasy kit (Qiagen) as described previously ( ).

Techniques: Binding Assay, Incubation, Concentration Assay, Labeling, Radioactivity, Inhibition, Standard Deviation, Infection, Amplification, Polymerase Chain Reaction, Generated, Clone Assay

Journal: Biochimica et biophysica acta

Article Title: Conformational change of the N-domain on formation of the complex between the GTPase domains of Thermus aquaticus Ffh and FtsY

doi:

Figure Lengend Snippet: Ffh/FtsY NG complex formation demonstrated by gel filtration chromatography. The 100-µl reactions were prepared with 10 µM FtsY, 15 µM Ffh NG and 1 mM GMPPCP in 50 mM HEPES (pH 7.5), 2 mM MgCl 2 , 50 mM NaCl and were incubated for the indicated time interval and temperature. The full reaction mixture was injected onto a Superdex 200 HR 10/30 column equilibrated with 50 mM HEPES (pH 7.5), 2 mM MgCl 2 , 50 mM NaCl. HPLC traces (280 nm) correspond to: (a) 42 °C overnight, (b) 3 days 37 °C, and (c) 4 days at 37 °C. No complex was observed after short incubations (1 h to overnight) over temperatures ranging from RT to 70 °C. Results from control reactions (all carried out at 37 °C, 4 days) are shown: (d) FtsY alone with GMPPCP, (e) Ffh/FtsY complex reaction with GDP substituted for GMPPCP; and (f) the reaction mixture plus 2 mM EDTA. The position of the complex peak is indicated (asterisk), and the elution volumes of 29 and 150 kDa standards are shown. The expected ratio of the total absorbance at 280 nm for the complex peak relative to the monomer peak assuming 100% complex yield under these conditions is ~ 8.3–1.

Article Snippet: Protease Inhibitor Cocktail containing 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) (at 0.2 mM final concentration), trans -epoxysuccinyl- l -leucylamido(4-guanidino) butane (E-64) (0.14 µM), bestatin (0.13 µM), leupeptin (0.1 µM), aprotinin (0.03 µM) and sodium EDTA (0.1 mM) was obtained from Sigma.

Techniques: Filtration, Chromatography, Incubation, Injection, High Performance Liquid Chromatography

Journal: eLife

Article Title: Type XVII collagen coordinates proliferation in the interfollicular epidermis

doi: 10.7554/eLife.26635

Figure Lengend Snippet: COL17 distribution is modulated by aPKC. ( a ) COL17 staining of whole IFE skin treated with 5 mM EDTA. The quantitative fluorescent intensity of COL17 in basal cells of IFE from control (PBS) and 5 mM EDTA treated (n = 4). Mann-Whitney test. Samples were taken from young adult WT mice at 6–10 weeks. Scale bar: 20 μm. ( b ) Phospho-aPKC labeling (indicated with arrows) and quantitative fluorescent intensity results from young and aged WT IFE skin (n = 4). Mann-Whitney test. Scale bar: 20 μm.( c ) Representative figures of asymmetric cell division (ACD; scored as perpendicular to basement membrane) and symmetric cell division (SCD; in parallel to basement membrane) in young IFE. Survivin staining indicates the direction of the cell division. Laminin β1 signifies basement membrane. Scale bar: 10 μm. Graph of percentage of ACD and SCD in young and aged IFE (n = 4). Student’s t-test. ( d–e ) The pharmacological inhibition of pan-aPKC (d, 1 μM of Go6983; 0.00002% DMSO as control) and aPKCλ/ζ (e, 10 μM of myr PSI; water as control) in whole IFE skin from young adult WT mice, followed by COL17 staining. The quantitative fluorescent intensity of COL17 in lateral membrane of basal cells from control and 1 μM Go6983 treated ( d ) and 1 μM myr PSI ( e ) (n = 4). Mann-Whitney test. Scale bar: 10 μm. BM, basement membrane. ( f–h ) EDTA treatment (5 mM; PBS as control, f ) and pharmacological inhibition of pan-aPKC (g, 1 μM of Go6983; 0.00002% DMSO as control) and aPKCλ/ζ (h, 10 μM of myr PSI; water as control) on 3D epidermis. The relative fluorescent intensity of COL17 in lateral membrane of basal cells was measured (n = 4). Mann-Whitney test. BM, basement membrane. Scale bar: 20 μm. The data are the means ± SE. *0.01

Article Snippet: Ex vivo inhibitor treatment Whole mice paw skin samples were treated with the following inhibitors for 1 hr at 4°C: EDTA, pan-PKC inhibitor Go6983 (Tocris Bio, Bristol, UK) and myristoylated pseudosubstrate Inhibitor (myr PSI; Calbiochem, Billerica, Massachusetts, USA) before staining.

Techniques: Staining, MANN-WHITNEY, Mouse Assay, Labeling, Inhibition