FIG. 1.

Effect of LEF-A titration on BKV load and RPTEC cytotoxicity. RPTECs (Lonza) (passage 4) were seeded in 24- or 96-well plates and supernatant infected with BKV-Dunlop at 50% confluency from Vero cells (multiplicity of infection [MOI] of 1) or left uninfected. At 2 h.p.i., virus or supernatant was removed, cells were washed, and medium with increasing LEF-A concentrations (A771726; Calbiochem) or without LEF-A was added. (A) Supernatants were harvested 72 h.p.i., and extracellular BKV loads were measured by qPCR with primers and probe targeting the LT-ag gene (5). Data are presented as Geq/ml (Geq = genome equivalents). (B) The cytotoxicity of LEF-A was monitored 72 h.p.i. by measuring cellular DNA replication by cell proliferation enzyme-linked immunosorbent assay (ELISA), BrdU (Roche Applied Science) (5), mitochondrial metabolic activity with cell proliferation reagent WST-1 (Roche Applied Science) (5), and total cellular metabolic activity (monotoring mithochondrial, microsomal, and cytosolic enzymes) with the resazurin-based assay TOX-8 (Sigma-Aldrich). For all three assays, colorimetric measurements were performed as described by the manufacturer. Absorbance for untreated cells was set as 100%.