Journal: Separations

Article Title: Alternative Method for HDL and Exosome Isolation with Small Serum Volumes and Their Characterizations

doi: 10.3390/separations8110204

Figure Lengend Snippet: Figure 6. The protein pattern and biomarker of the exosomes isolated from different sample volumes. A protein ladder reference (Vivantis Technologies, Shah Alam, Selangor Darul Ehsan, Malaysia, cat no. PR0623) had 2 reference bands (25 and 72 kDa) coupled with blue chromophore for easy identification (A). SDS-PAGE stained with Coomassie blue G250 showed multiple bands with the same patterns (B). The Western blot results showed a single band of TSG101 at about 48.9 kDa exhibiting the exosome biomarker (C). Thirty micrograms of protein were loaded for both SDS-PAGE and the Western blot. All experiments above were run with a reducing agent. M = Marker of protein molecular weight.

Article Snippet: The primary antibodies, human ApoA-1 antibody (cat no. MAB 36641-SP) and human TSG101 antibody (cat no. orb337267), were supplied by R&D System, Inc., Minneapolis, MN, USA and Biorbyt, Cambridge, Cambridgeshire, UK, respectively.

Techniques: Biomarker Discovery, Isolation, SDS Page, Staining, Western Blot, Marker, Molecular Weight

Journal: PLoS Pathogens

Article Title: ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus

doi: 10.1371/journal.ppat.1005960

Figure Lengend Snippet: List of antibodies used in the study and their source.

Article Snippet: The siRNA oligonucleotides against Tsg101 (pool of 3 target specific siRNAs) were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.

Techniques:

Journal: PLoS Pathogens

Article Title: ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus

doi: 10.1371/journal.ppat.1005960

Figure Lengend Snippet: (A) HMVEC-d cells were transfected with siControl or siTsg101-RNA and examined after 48 h by Western blotting for Tsg101 and β-Tubulin. (B, C) siControl and siTsg101-RNA transfected HMVEC-d and HUVEC cells were infected with KSHV (30 DNA copies/cell) for 2 h at 37°C, washed and treated with 0.25% trypsin-EDTA for 5 min to remove the bound and non-entered virus. The cells were then treated with DNAse I for 10 min at 37°C according to the manufacturer's protocol to remove any contaminating DNA and assess only the viral DNA that was internalized. Total DNA was extracted and entry was determined by real-time DNA PCR for the KSHV ORF73 gene. The percentage of KSHV entry was calculated by considering the siControl value as 100%. Each reaction was done in triplicate and results presented are means ± SD of three independent experiments. (D) siControl and siTsg101-RNA transfected HMVEC-d cells were infected with BrdU (viral DNA) labeled KSHV (30 DNA copies/cell) for 30 min at 37°C, washed, treated with trypsin-EDTA for 5 min at 37°C, fixed and permeabilized using 0.2% Triton X-100 in PBS for 5 min, washed and followed by a denaturation step with 4N HCl for 10 min at RT to expose incorporated BrdU residues, washed, reacted with anti-BrdU antibodies followed by Alexa 488-anti-mouse antibodies and examined by immunofluorescence assay (IFA). Representative images are shown. White and red arrows indicate BrdU-KSHV genome associated with infected cell nuclei and cytoplasm, respectively. Magnification, 40x. (E) The percentage of nuclei positive for BrdU staining is presented graphically. A minimum of five independent fields, each with at least 10 cells was chosen. Results presented are means ± SD of three independent experiments. **p<0.01. (F, G) siControl and siTsg101-RNA transfected HMVEC-d and HUVEC cells were infected with KSHV (30 DNA copies/cell) for 2 h at 37°C, washed, treated with 0.25% trypsin-EDTA for 5 min at 37°C, washed, treated with DNAse I for 10 min at 37°C, washed, lysed on ice for 5 min with a mild lysis buffer, centrifuged at 500xg for 5 min to obtain the concentrated nuclear pellet. The cytoskeletal components loosely bound to the nuclei were removed by a second cycle of lysis and centrifugation steps . The pure nuclear fraction was then used for DNA isolation and nuclear entry of the KSHV genome was determined by real-time DNA-PCR for the KSHV ORF73 gene. The percentage nuclear entry of genome was calculated considering the siControl value as 100%. Each reaction was done in triplicate. Results presented are means ± SD of three independent experiments. **p<0.01.

Article Snippet: The siRNA oligonucleotides against Tsg101 (pool of 3 target specific siRNAs) were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.

Techniques: Transfection, Western Blot, Infection, Virus, Labeling, Immunofluorescence, BrdU Staining, Lysis, Centrifugation, DNA Extraction

Journal: PLoS Pathogens

Article Title: ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus

doi: 10.1371/journal.ppat.1005960

Figure Lengend Snippet: (A) HMVEC-d cells were left uninfected or infected with 30 DNA copies/cell of KSHV at different time points as indicated, washed, fixed, permeabilized, blocked, stained for KSHV-gB, co-stained for Tsg101 and examined by immunofluorescence microscopy. Magnification, 40x. Boxed areas are enlarged in the rightmost panels. White arrows indicate colocalization between KSHV-gB and Tsg101. (B) HMVEC-d cells were infected with KSHV (30 DNA copies/cell) for 10 min, washed and immunoprecipitated with anti-Tsg101 antibody and analyzed for the KSHV glycoprotein gB by western blot. 20 μg of whole cell lysate protein was also used to analyze the total protein levels. (C) HMVEC-d cells transfected with control or siTsg101-RNA were infected with KSHV (30 DNA copies/cell) followed by IFA using anti-KSHV gpK8.1A antibody and rhodamine phalloidin for F-actin. Representative images are shown. Magnification, 80x. White arrows indicate KSHV gpK8.1A.

Article Snippet: The siRNA oligonucleotides against Tsg101 (pool of 3 target specific siRNAs) were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.

Techniques: Infection, Staining, Immunofluorescence, Microscopy, Immunoprecipitation, Western Blot, Transfection, Control

Journal: PLoS Pathogens

Article Title: ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus

doi: 10.1371/journal.ppat.1005960

Figure Lengend Snippet: (A) HMVEC-d cells were left uninfected or infected with 30 DNA copies/cell of KSHV and Texas red labeled dextran (40-kDa, 0.5 mg/ml) at 37°C for the indicated time points. The cells were washed, fixed, permeabilized, blocked, and processed for triple IFA using anti-Tsg101 antibodies (blue) and anti-gB (green) antibodies, and dextran (red). Representative images are shown. Magnification, 80x. Boxed areas are enlarged in the rightmost panels. Yellow arrows indicate triple colocalization. (B) A minimum of 10 cells were analyzed in 5 different optical regions to calculate the averages of the colocalization. The colocalizations of mean pixel intensities were analyzed with the Nikon NIS-Elements AR imaging software (Nikon). Mean colocalization pixel intensities were measured in arbitrary units (a.u.) and are presented as the percent colocalization. Error bars show ± SD. ***p<0.001. (C) A schematic diagram summarizing the results of our previous studies [ , , ] to show the KSHV interaction with receptors, stages of bleb formation that gradually lead to macropinocytosis of KSHV and trafficking into the HMVEC-d cell. The doted box is enlarged to show the virion particle in the macropinosome/early endosome (Rab5+) interacting with the internalized receptors in the vesicle side and the receptors and the associated signal molecules in the cytoplasmic side of the macropinosome.

Article Snippet: The siRNA oligonucleotides against Tsg101 (pool of 3 target specific siRNAs) were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.

Techniques: Infection, Labeling, Imaging, Software

Journal: PLoS Pathogens

Article Title: ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus

doi: 10.1371/journal.ppat.1005960

Figure Lengend Snippet: (Ai, ii, iii) Serum-starved (8 h) HMVEC-d cells were either mock or KSHV infected (30 DNA copies/cell) for the indicated time points, washed, immunoprecipitated with anti-Tsg101 antibodies and analyzed by Western blot (WB) for EphA2R, c-Cbl, p130Cas, Crk and Tsg101 molecules (as indicated). 20 μg of whole-cell lysate (WCL) protein was analyzed by Western blot to check for total protein levels. Actin was used as loading control. ( B, D, F, H ) Proximity ligation assay (PLA) [ , , ]. HMVEC-d cells, infected with KSHV for 10 min at 37°C were washed and processed for PLA using oligonucleotide conjugated PLA probes. The PLA signals indicating the association of Tsg101 with several signal molecules were observed as green dots by fluorescent microscopy. The nuclear staining was performed with DAPI. Magnification, 40x. (C, E, G, I) The quantification of interaction (colocalization) of Tsg101 with the respective signal proteins is presented graphically as the average interacting PLA dots per cell. A minimum of five independent fields, each with at least 10 cells, were chosen. Error bars show ± SD. **p<0.01, ***p<0.001.

Article Snippet: The siRNA oligonucleotides against Tsg101 (pool of 3 target specific siRNAs) were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.

Techniques: Infection, Immunoprecipitation, Western Blot, Control, Proximity Ligation Assay, Microscopy, Staining

Journal: PLoS Pathogens

Article Title: ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus

doi: 10.1371/journal.ppat.1005960

Figure Lengend Snippet: Serum starved HMVEC-d cells were infected with KSHV (30 DNA copies/cell) for the indicated time points, washed, and processed for IFA and PLA. Nuclei were stained with DAPI and representative images are shown. Magnification, 40x. Boxed areas from the merged panels are enlarged in the rightmost panels. (A) IFA with mouse anti-Tsg101 (green) and rabbit anti-Rab5 (red) antibodies. White arrows indicate colocalization of Tsg101 with Rab 5 (yellow). (B) Graphical representation of the colocalization shown in 6A as per methods described under the legend. (C) PLA assay using mouse anti-Tsg101 and rabbit anti-Rab5 antibodies. White arrows indicate red spots representing Tsg101 in close proximity with Rab5 positive vesicles. (D) Graphical representation of the average Tsg101-Rab 5 interaction PLA dots per cell shown in 6C. (E) Western blot analysis showing no change in Rab5 protein level upon KSHV infection at the indicated time points.

Article Snippet: The siRNA oligonucleotides against Tsg101 (pool of 3 target specific siRNAs) were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.

Techniques: Infection, Staining, Western Blot

Journal: PLoS Pathogens

Article Title: ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus

doi: 10.1371/journal.ppat.1005960

Figure Lengend Snippet: Serum starved HMVEC-d cells were infected with KSHV (30 DNA copies/cell) for the indicated time points, washed, and processed for IFA and PLA. Nuclei were stained with DAPI and representative images are shown. Magnification, 40x. (A) IFA with mouse anti-Tsg101 and rabbit anti-Rab7 antibodies. White arrows indicate colocalization of Tsg101 with Rab 7 (yellow). (B) Graphical representation of the colocalization shown in 7A as per methods described under the legend. (C) PLA using mouse anti-Tsg101 and rabbit anti-Rab7 antibodies. White arrows indicate red spots representing Tsg101 in close proximity with Rab7 positive vesicles. (D) Graphical representation of the average Tsg101-Rab 7 interaction PLA dots per cell shown in 7C. (E) Western blot analysis showing no change in Rab7 protein level upon KSHV infection at the indicated time points.

Article Snippet: The siRNA oligonucleotides against Tsg101 (pool of 3 target specific siRNAs) were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.

Techniques: Infection, Staining, Western Blot

Journal: PLoS Pathogens

Article Title: ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus

doi: 10.1371/journal.ppat.1005960

Figure Lengend Snippet: (A) A schematic representation of the ESCRT complex proteins showing what is known about the sequential recruitment of proteins on the endosomal membrane followed by formation of the multi-vesicular body. (B) Serum-starved (8 h) HMVEC-d cells were either mock or KSHV infected (30 DNA copies/cell) for the indicated time points, immunoprecipitated with anti-Tsg101 antibodies and analyzed by Western blot for other interacting ESCRT complex proteins (as indicated). 20 μg of whole-cell lysate protein was analyzed by Western blot to check for total protein levels of Tsg101. β-tubulin was used as loading control. ( C, E, G, I ) PLA reactions. HMVEC-d cells infected with KSHV for 10 min at 37°C were washed and processed for PLA and signals indicating the association of Tsg101 with other molecules were observed as red or green dots by fluorescent microscopy. The nuclear staining was performed with DAPI. Magnification, 40x. (D, F, H, J) The quantification of interaction (colocalization) of Tsg101 with other ESCRT complex proteins is presented graphically as the average interacting PLA dots per cell. A minimum of five independent fields, each with at least 10 cells were chosen, and the error bars show ± SD. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The siRNA oligonucleotides against Tsg101 (pool of 3 target specific siRNAs) were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.

Techniques: Membrane, Infection, Immunoprecipitation, Western Blot, Control, Microscopy, Staining

Journal: PLoS Pathogens

Article Title: ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus

doi: 10.1371/journal.ppat.1005960

Figure Lengend Snippet: Serum starved control siRNA or Tsg101 siRNA transfected HMVEC-d and HUVEC cells infected with KSHV for 10 min at 37°C were washed and processed for IFA. Magnification, 40x. Representative deconvoluted images are shown. The white boxes within the merged panels are shown as enlarged pictures and the white arrows represent colocalization of the indicated molecules. (A, B) IFA with rabbit anti-Rab5 and mouse anti-gpK8.1A antibodies. (E, F) IFA with Rabbit anti-Rab7 and mouse anti-gpK8.1A antibodies. These were followed by staining with secondary antibodies conjugated to Alexa fluor 594 (red) and Alexa fluor 488 (green). (C, D, G, H) Percentage colocalizations shown in figure A, B, E and F, respectively, were calculated by analyzing a minimum of 10 cells in 5 different optical regions as per methods described under the legend. Error bars show ± SD. ***p<0.001.

Article Snippet: The siRNA oligonucleotides against Tsg101 (pool of 3 target specific siRNAs) were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.

Techniques: Control, Transfection, Infection, Staining

Journal: PLoS Pathogens

Article Title: ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus

doi: 10.1371/journal.ppat.1005960

Figure Lengend Snippet: KSHV entry into HMVEC-d cells begins with (1) the virus attachment to the cell surface heparan sulfate followed by sequential interactions with α3β1, αVβ3 and αVβ5 integrins, and the CD98-xCT molecule, rapid induction of host cell FAK, Src, PI3-K, RhoGTP and ROS signal molecules, PI3-K mediated activation of cCbl which monoubiquitinates the α3β1 and αVβ3 integrins resulting in the rapid lateral translocation of virus-bound α3β1 and αVβ3 integrins into the lipid raft (LR) regions of the plasma membrane. LR translocated KSHV interacts with and activates LR-associated Ephrin A2 receptor tyrosine kinase (EphA2R), which results in the recruitment of the c-Cbl-integrin-myosin IIA light chain complex to the LR region and amplification of Src, PI3-K and c-Cbl activation and recruitment of CIB1-p130Cas-Crk molecules to EphA2R that are crucial for macropinocytosis. (2) The interaction of activated c-Cbl with myosin IIA leads to bleb formation and its retraction leading to macropinosome (early endosome) formation. (3) Simultaneously, EphA2R and CIB1-p130Cas-Crk coordinated signal amplification and cytoskeletal crosstalk results in the association with ESCRT-0 Hrs protein to the site of macropinocytosis, ROCK1 activation, and phosphorylation of NHE1, an intracellular pH-regulating protein, a local membrane pH change required for macropinocytosis. The present studies show the (4) sequential recruitment of ESCRT-I Tsg101 protein and several ESCRT I-III complex proteins on the early and late endosome. The Tsg101 molecule associates with the KSHV containing macropinosomes, and increased levels of Tsg101 associates/interacts with EphA2R, c-Cbl, p130Cas and Crk signal molecules, upstream and downstream ESCRT components Hrs (ESCRT-O), EAP45 (ESCRT-II), CHMP6 (ESCRT-III) and CHMP5 (ESCRT-III) in the KSHV infected cells, and early (Rab5) and late endosomal (Rab7) stages of KSHV intracellular trafficking. This also becomes the limiting step in KSHV’s transition from the early to late endosome in the absence of Tsg101 protein. (5) The KSHV lipid envelope membrane fuses with the endosomal membrane mediated by the viral glycoproteins and perhaps by other host proteins, to release the capsid and the enclosed viral genome into the cytoplasm. (6) Viral genome enters the nucleus and viral gene expression occurs with the help of KSHV infection induced host signaling molecules such as NF-kB, ERK1/2, and Nrf2 [ – ].

Article Snippet: The siRNA oligonucleotides against Tsg101 (pool of 3 target specific siRNAs) were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.

Techniques: Virus, Activation Assay, Translocation Assay, Clinical Proteomics, Membrane, Amplification, Phospho-proteomics, Infection, Gene Expression

Journal: Nature Communications

Article Title: Plk1 overexpression induces chromosomal instability and suppresses tumor development

doi: 10.1038/s41467-018-05429-5

Figure Lengend Snippet: Overexpression of Plk1 impairs Cep55 and ESCRT loading into the cytokinesis midbody. a (+/Plk1);(rtTA/rtTA) MEFs were untreated (–Dox) or treated for 24 h with doxycycline (+Dox), fixed and stained for α-tubulin (red) and DAPI (DNA, green). Cells in cytokinesis ( n > 100 per condition; n = 3 replicates) were evaluated for aberrant cytokinesis, considering the midbody formation and shape, and correct distribution of DNA into the two daughter cells. Scale bars, 10 μm. b The length of the cytokinesis bridge was evaluated by measuring the distance in between the two daughter nuclei in MEFs untreated or after 24 h of Dox treatment (–Dox, n = 17 cells; +Dox, 23 cells). Scale bars, 10 μm. In a , b *, p < 0.05; Student’s t -test. c MEFs expressing a CEP55-EGFP fusion (green) were treated for 24 h with Dox (+Dox), in the absence or presence of 1 μM of the Plk1 inhibitor BI2536 for 1 h at the end of the Dox time (Dox + BI), or left untreated as control (–Dox). α-tubulin is in red. Data represent the percentage of cells with positive CEP55-EGFP signal at the midbody (–Dox, n = 134; +Dox, n = 94; Dox + BI, n = 47 cells; n = 3 replicates (–Dox, +Dox) or 2 replicates (Dox+BI)). Scale bar, 5 μm. d MEFs expressing a Tsg101-mCherry fusion (red) were treated for 24 h with Dox (+Dox), in the absence or presence of 1 μM of BI2536 for 1 h at the end of the Dox time (Dox + BI), or left untreated as control (–Dox). α-tubulin is in green. Data represent Tsg101-mCherry mean intensity at the midbody (–Dox, n = 81; +Dox, n = 60; Dox + BI, n = 37 cells; n = 2 replicates). Scale bars, 5 μm. e MEFs were treated for 24 h with Dox (+Dox), in the absence or presence of 1 μM of BI2536 for 1 h at the end of the Dox time (Dox + BI), or left untreated (–Dox). Cells were stained for α-tubulin (red) and DAPI (DNA, green) and binucleation index was quantified from more than 600 cells in each sample ( n = 5 replicates). Scale bars, 50 μm. In c – e , n.s. not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; one-way ANOVA

Article Snippet: MEFs were transfected either with cDNA encoding for MKLP1 fused to GFP (Addgene #70145), CEP55 (obtained from the Mammalian Gene Collection) fused to GFP, or the ESCRT-I member TSG101 fused to the mCherry reporter (Addgene #38318).

Techniques: Over Expression, Staining, Expressing, Control