Figure 10.

FGF-2, alone or combined with Tat, increased the proliferation and survival of cultured podocytes. (A) Proliferation assay. Podocytes were seeded at a density of 5×10⁴ cells/well and cultured in DMEM media supplemented with 1% FBS and penicillin/streptomycin for 4 days. HIV-Tat, FGF-2, or both combined were added daily at a concentration of 50 ng/ml each. On day 4, all cells were trypsinized and counted as described in Concise Methods. Values significantly different from controls are marked: *P<0.05. (B) The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) survival assay. Podocytes were seeded at a density of 6×10⁴ cells per well and starved overnight on DMEM serum-free media containing antibiotics. Subsequently, all cells were treated with FGF-2+Tat at a concentration of 10 ng/ml each (black bars) in the presence or absence of the corresponding inhibitors. The kinase inhibitor PD98059 (5 µM), the Rho-associated protein kinase inhibitor Y27632 (10 µM), and the Rho-A inhibitor C3-transferase (20 ng/ml) were added 2 hours before the FGF-2+Tat treatment. On day 3, 10 µl MTT solution (5 mg/ml) was added to each well, incubated for 3 hours at 37°C, and then, treated with 100 µl MTT solvent (4 mM HCl and 0.1% Triton X-100 in isopropronal) as described in Concise Methods. Results were recorded in MTT absorbance units and expressed as a percent of control values (open bars) considering six independent readings. Values significantly different from controls are marked: **P<0.01.