Fat free bsa

Human podocytes differentiated for 13 days were labeled for 24 h at 37 °C in RPMI medium containing 2% FBS and [³H]-cholesterol (1 μCi/ml, American Radiolabeled Chemicals). Cells were then washed 3 times with PBS and incubated with RPMI supplemented with 0.2% fat free-BSA (Sigma) with or without compounds for 18-h at 37 °C. The compounds were used at the following concentrations: Cpd C (1 μM), Cpd A (1 μM, 5 μM) and Cpd G (1 μM, 5 μM, 10 μM). Following the 18-h incubation, human apoAI (Calbiochem) was added to the media, (20 μg/mL final concentration) and the cells were incubated for another 18 h at 37 °C. Aliquots of medium collected before (T  = 0 h) and after (T = 18 h) the addition of apoAI were centrifuged at 12,000 × g for 5 min and the radioactivity in the supernatant was counted by liquid scintillation. Cells were then washed with PBS, lysed 0.1% SDS, 0.1M NaOH and the radioactivity in the lysates was quantified by liquid scintillation. apoAI-mediated cholesterol efflux, expressed as %, was calculated as the amount of label released to the medium after adding apoAI (the difference in radioactivity in the medium before and after adding ApoA1) divided by the amount of total label in each well (radioactivity released to the media plus radioactivity in the lysed cells).

To analyze the cellular fate of LDL-cholesterol, cells were plated on coverslips and grown in F-12 (HAM) supplemented with 5% LPDS for 48 h. Cells were then loaded with 50 µg/µl of LDL ± 10 µg/ml ACAT inhibitor (Sandow 58-035) for 24 h, fixed with 4% PFA for 1 h. Free cholesterol was stained with 0.05 mg/ml of filipin (Sigma Aldrich) and neutral lipids were stained with 1 µg/ml of BODIPY 493/503 (Molecular Probes) for 20 min at RT. Coverslips were mounted in Mowiol (Calbiochem, Merck). Alternatively, cellular cholesterol was stained with recombinant GST–PFO as follows: cells were fixed with 4% PFA for 15 min, permeabilized with 0.1% Triton X-100 (Sigma Aldrich) for 5 min and blocked with 3% fat free BSA (Sigma Aldrich) PBS for 30 min at RT. Cells were incubated with 10 µg/ml of purified recombinant GST–PFO in blocking buffer for 1 h at RT. Immunostaining with anti-GST (Abcam) and fluorescently labeled antibody was performed as above.