Fig. 5. Cholesterol content is increased in LRP1 knockdown cells via increased cholesterol import.

(A) Reduced LRP1 activity increases intracellular cholesterol: Fibroblasts were untreated, transfected with LRP1-specific or scrambled control siRNA for 24 h, or treated with LRP1 or tubulin antibody for 1 h; and then cells were mock infected or infected (5 IU/cell). At 24 hpi, cell cholesterol content was quantified.

(B) Inhibition of cholesterol synthesis had a modest effect, whereas extracellular cholesterol was needed to increase cellular cholesterol after LRP1 inhibition: Cholesterol was quantified after treatment with simvastatin (2.5 µM) or LDL-cholesterol (50 µg/ml) at 24 hpi.

(C) Inhibition of cholesterol synthesis does not block the enhanced yield of infectious virus produced by inhibition of LRP1: Fibroblasts were untreated, treated with simvastatin (2.5 µM), or mock treated; and 30 min later, cells were treated with antibody to LRP1 or tubulin or left untreated for 1 h. Cells were subsequently infected (0.1 IU/cell). At 96 hpi, released virus was quantified by TCID₅₀ assay. (D) Extracellular cholesterol is required for the enhanced yield of infectious virus produced by inhibition of LRP1: Cells were serum starved for 48 h, treated first with the indicated antibodies for 1 h and then with various concentrations of LDL-cholesterol for 1 h. Next, cells were infected (0.1 PFU/cell) and released virus was assayed by TCID₅₀ assay at 96 hpi.

Error bars report the standard error from three experiments with three replicates each. *P<0.001 (t-test, compared to equivalent control condition). Fig. S2 contains additional data relevant to this experiment.