Figure 3
siRNA knockdown of Cav-1 and its effect on cholesterol of cells expressing wild type Nef or NefG2A. (A). U87-CD4-CXCR4 cells were transfected with Cav-1 siRNA or control siRNA (CTL-siRNA) followed by transfection with Nef expressing plasmid (pSHIVwtNef) or pCDNA3.1 vector. The level of Cav-1 expression in siRNA treated cells was detected by Western blotting analysis. Cells were labelled with [3H] cholesterol for 36 hours and apoA-I induced cholesterol efflux was measured. Results are shown as the percentage of cholesterol efflux relative to control. (B) THP-1 cell-differentiated macrophages were first transfected with Cav-1 siRNA or CTL-siRNA which was followed by infection with VSV-G pseudotyped HIV (psHIVwtNef) at an moi of 3. Cells were again treated with siRNA the day after infection. Cholestrol efflux was measuered as described above four days post-infection. (C) U87-CD4-CXCR4 cells were transfected with pCI (Mock), pCI NL4-3 Nef-HA-WT (Nef), pCI NL4-3 NefG2A-HA (NefG2A), or pCI NL4-3 NefG2A-HA along with pCZ-cav-1 (Nef plus Cav-1). Cholestrol efflux to apoA-I was determined. All experiments were performed in triplicate and results shown are mean ± SD with P values.