DMEM, trypsin and antibiotics were purchased from Mediatech Inc. (Manassas, VA, USA). Antibodies against CAV1 and cell culture inserts with transparent polyethylene terephthalate (PET) membranes with 3µm pores were from BD Biosciences (Franklin Lakes, NJ, USA). The antibody against the Myc tag and HRP-conjugated secondary antibodies were from Cell Signaling Technology (Danvers, MA, USA). Slide-A-Lyzer MINI Dialysis Units and the ECL Western Blotting substrate were purchased from Thermo Scientific (Rockford, IL, USA). Lipofectamine 2000 and Alexa Fluor 488-conjugated secondary antibody were from Invitrogen (Carlsbad, CA, USA). The protease inhibitor cocktail was from Roche Applied Science (Indianapolis, IN, USA), the CellTiter-Glo Luminescent Cell Viability Assay kit was from Promega (Madison, WI, USA), the 0.45µm MCE filter units were from Fisher Scientific (Pittsburgh, PA, USA), and a TrueORF Gold Myc-DDK-tagged human CAV1 plasmid was from Origene (Rockville, MD, USA). Anti-FLAG M2 Magnetic Beads were from Sigma-Aldrich (St. Louis, MO, USA) and Fluoro-Gel cell mounting medium was obtained from Electron Microscopy Sciences (Hatfield, PA, USA). Unless otherwise mentioned, most chemicals used in the study were of molecular biology or cell culture grade and were procured from either Fisher Scientific or Sigma-Aldrich.
Cell culture and preparation of conditioned media
The EWS cell lines (A4573, SK-ES-1 and TC-71) and the prostate cancer cell line (PC3) were maintained in DMEM basal medium supplemented with antibiotics and 10% fetal bovine serum, and is henceforth referred to as the “culture medium”. Cells were incubated at 37 °C in a humidified atmosphere with 5% CO2
. The CAV1 knocked-down A4573/shCAV1 cells were generated and maintained as described earlier (7
). Conditioned media were prepared by collecting the culture fluids from sub-confluent cultures and clearing the cell debris by centrifugation at 1,000 ×g
, followed by filtration through 0.45 µm MCE filter units.
CAV1 from cell lysates or conditioned media was detected using either dot-blot assays or western blotting. Cells were lysed with RIPA buffer containing protease inhibitor cocktail (Roche), and the lysates were centrifuged at 13,000 ×g, at 4 °C, for 15 min. Proteins obtained from lysed cells were resolved by 4–20% SDS-PAGE, and transferred to PVDF membranes. To confirm the secretion of CAV1, different volumes of conditioned media from cultures of EWS cell lines and PC3 cells were spotted on PVDF membranes, using a dot-blotting apparatus.
CAV1 immunodetection in media by western blotting was carried out by concentrating the conditioned media through precipitation with either trichloroacetic acid (TCA) or ammonium sulfate. TCA (10% by vol) was added to 1 ml of conditioned media, incubated for 30 min on ice and centrifuged for 30 min at 10,000 ×g. The pellet was washed in acetone, air dried and resuspended in 100 µl of SDS sample buffer. Proteins present in the pellet (30 µl) were resolved by 4–20% SDS-PAGE, and transferred to PVDF membranes.
For ammonium sulfate precipitation of proteins, conditioned media were centrifuged at 1,000 ×g for 15 min, at 4 °C, to remove debris. The supernatant collected was subjected to sequential 0–30, 30–70 and 70–90% ammonium sulfate fractionation, at 4 °C, maintaining the pH between 7.2 and 7.6. The ammonium sulfate precipitated fractions were resuspended in 150–200 µl of PBS and dialyzed in Slide-A-Lyzer MINI Dialysis Units against 1 liter of PBS for 16 h. The dialyzed samples were centrifuged at 10,000 ×g, at 4 °C, for 30 min, to remove any particulate matter, and the concentration of protein was measured. Equal amounts of protein were subsequently subjected to SDS/PAGE analysis to optimize the percentage of ammonium sulfate required for CAV1 precipitation from the media.
Following transfer, the PVDF membranes were blocked with 5% skim milk in PBST (PBS containing 0.2% Tween-20) at room temperature for 1 h and incubated overnight at 4 °C with designated primary antibodies, with gentle rocking. Next, the blots were washed with PBST and incubated at room temperature for 1 h with an HRP-conjugated secondary antibody and peroxidase activity was detected by enhanced chemiluminescence, using the ECL western blotting substrate.
Transfection of human CAV1
To express CAV1 in EWS cells, a validated TrueORF Gold Myc-DDK-tagged human CAV1 plasmid was transfected into EWS cells using Lipofectamine 2000 following manufacturer's protocols. Transfected cells were selected with G418 (1 mg/ml) for 14 days, and antibiotic-resistant colonies were pooled for further analysis and maintained in the presence of G418 (0.5 mg/ml).
Purification of Myc-DDK-CAV1
The DDK tag is a synonym for FLAG tag, hence Myc-DDK-tagged CAV1 expressed in A4573 and SK-ES-1 cells was purified using anti-FLAG magnetic beads according to the manufacturer’s protocol. Briefly, cells expressing the tagged protein were lysed at 4 °C for 15 min in lysis buffer containing 50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100, supplemented with the protease inhibitor cocktail. Lysates were centrifuged at 13,000 ×g, at 4 °C, for 15 min. About 300–400 µl of cell lysate were added to 40 µl of resin beads, and the volume was made up to 1 ml with the lysis buffer. The sample was incubated at room temperature on a roller shaker for 2 h and then placed on a magnetic separator to remove the supernatant. The beads were washed 5 times with TBS (50 mM Tris-Cl, 150 mM NaCl, pH 7.4), and bound protein was eluted with 50 µl of 0.1 M glycine HCl, pH 3.0. The eluted protein was neutralized with 15 µl of Tris-Cl, pH 8.0, and subsequently used for further studies. The Myc-DDK-tagged CAV1 is henceforth referred to as MD-CAV1.
A4573 cells were seeded on glass cover slips in 12-well plates and grown in 1 ml culture medium for 2 days. The culture medium was replaced with 400 µl of fresh medium in each well. Subsequently, three different sets were assigned for experimental purposes. In the first set, cell culture inserts with transparent PET membranes (with 3µm pores) were added to wells with cells on cover slips and 500 µl of culture-media containing 2 × 104 A4573 cells expressing MD-CAV1 were seeded on the membrane in the inserts. In the second set, purified MD-CAV1 was added in 500 µl of culture medium directly to the cells on cover slips, while in the third set fresh culture medium was added to the cells on cover slips. The cells were allowed to grow for 16 h, following which the cells on the cover slips were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and equilibrated in PBS. The cover slips with fixed cells were pre-incubated for 30 min in 2% BSA-PBS, followed by 1 h incubation with mouse anti-Myc antibody (1:5000 dilution in 1% BSA-PBS) at room temperature. The cover slips were washed thrice with PBS and incubated for 45 min with Alexa Fluor 488 conjugated secondary antibody (1:2000 dilutions in 1% BSA-PBS). The cells were washed twice with PBS, before incubating them with DAPI for 30 min in the dark. Finally, the cells were washed twice in PBS and once in deionized water before being mounted in Fluoro-Gel mounting medium. Images were recorded in an Olympus microscope and analyzed using software provided by the manufacturer.
Cell proliferation assays
Cell proliferation was measured using the CellTiter-Glo Luminescent Cell Viability Assay kit, according to the manufacturer’s protocol in a 96-well plate format. Equal number of cells were seeded in 96-well plates and following 24 h of growth were subjected to the following conditions: (i) culture-medium (control); (ii) 1:1 CAV1 conditioned medium and culture-medium; (iii) culture medium with purified MD-CAV1; (iv) culture-medium with purified MD-CAV1 plus anti-CAV1 antibodies; and (v) culture-medium with anti-CAV1 antibodies only. Following growth for 48 h, the luminescence intensity measured for each sample was plotted relative to the values obtained for the control cells. The data represent the average from at least 3 independent experiments.
Significance (P)-values between dataset pairs were obtained from two-tailed Student's t-tests, and the data presented as the average with the standard deviation as error bars.