Effect of cholesterol depletion and cholesterol-loading on TF functional expression and reversibility of cholesterol effect. (A-C) Monolayers of WI-38 cells were treated for 45 minutes at 37°C with mβCD (10 mM) to deplete cholesterol or water-soluble cholesterol (mβCD-cholesterol, 1 mM) to load the cells with cholesterol. Then, the monolayers were incubated with (A) unlabeled VIIa (10 nM), followed by substrate factor X (175 nM) for 5 minutes at 37°C to measure TF functional activity; (B) ¹²⁵I-VIIa (10 nM) or (C) ¹²⁵I-TF mAB for 1 hour to measure VIIa or TF mAB binding to the cells. (D-E) Monolayers were first treated for 30 minutes at 37°C with mβCD (10 mM) to deplete cholesterol. After washing monolayers, cholesterol was reintroduced to the cells by incubating the cholesterol-depleted cells with cholesterol (1 mM): mβCD (10 mM) for 30 minutes. Following this, the cells were washed with buffer B and used to determine cell surface TF activity (D) and ¹²⁵I-VIIa binding to cell surface TF (E) (n = 3, mean ± SE). * denotes significantly (P < .05) differs form the control; # denotes significantly (P < .05) differs from both the control and the cholesterol-depleted cells; and ** denotes significantly (P < .05) differs from mβCD-treated cells but not from the control. In A-C, ▩ indicates control; ▪, cholesterol-deplete; ▨, cholesterol-laden. In D and E, ▩ indicates control; ▪, mβCD; and ▨, mβCD plus cholesterol.