Induction of cholesterol efflux attenuates the enhancement in HIV-1infectivity in NPCD55 cells. (A) Normal and NPCD55 cells were treated with 5 μM TO-901317 at 24 h post-infection and cultured for 72 h in the presence of the compound. At 96 h post-infection the cells were harvested and ABCA1, Gag, and β-actin expression was detected by Western blotting analysis. All samples were loaded on the same gel. (B) AmplexRed assay was performed to measure free cholesterol content of untreated (filled) and TO-901317-treated (slashed) cells. Cholesterol content was normalized to protein concentration. (C) AmplexRed assay was performed to measure virion-associated cholesterol from purified virus produced in untreated (filled) and TO-901317-treated (slashed) infected cells. Virion-associated cholesterol content was normalized to p24 concentration. (D) TZM-bl reporter assay was performed to measure virus infectivity when cholesterol efflux was induced via TO-901317 stimulation. The ρ values were calculated by performing student T-test (*denotes < 0.05).