Fig. 4

Effect of M-Sec knockdown on quantity of HIV-1 in cellular fraction of U87 cells. a U87.CD4.CCR5 cells were transfected with either control (Cr pool #2) or M-Sec-specific siRNA (#4) and cultured for 2 days. Cells were infected with JRFL-GFP (input: 100 ng/ml p24 Gag), cultured for 1–2 days, and analyzed for GFP-positive cell percentage by flow cytometry (mean ± SD, n = 3). n.s. not significant. *p < 0.05. dpi, days postinfection. b U87.CD4.CCR5 cells were transfected and infected as in (a), and cultured for 2 days and analyzed for GFP expression by flow cytometry. Mean fluorescence intensity (MFI) in the GFP-positive fraction is shown (mean ± SD, n = 3). n.s. not significant. dpi, days postinfection. c U87.CD4.CCR5 cells were transfected with either control (Cr pool #2) or M-Sec-specific siRNA (#4) and cultured for 2 days. Cells were infected with JRFL (input: 100 ng/ml p24 Gag), cultured for 2 days, and analyzed for Env expression by immunofluorescence. The Env signal was quantified in three different fields for each group, and the mean intensity of the Env signal per field is shown. *p < 0.05. n.s. not significant; dpi, days postinfection. d U87.CD4.CCR5 (left) or U87.CD4.CXCR4 cells (right) were transfected with either control (Cr pool #2) or M-Sec-specific siRNA (#4), cultured for 2 days, and analyzed for CD4 expression by flow cytometry. The mean fluorescence intensity (MFI) of CD4 is shown (mean ± SD, n = 3). n.s. not significant. e U87.CD4.CCR5 (upper) or U87.CD4.CXCR4 cells (lower) were transfected with either control (Cr pool #2) or M-Sec-specific siRNA (#4), and cultured for 2 days. U87.CD4.CCR5 and U87.CD4.CXCR4 cells were infected with JRFL (input: 100 ng/ml p24 Gag) and NL43 (input: 1 ng/ml p24 Gag), respectively, and cultured for 5 days. Total cell lysates were prepared at day 0, 2, or 5, and subjected to western blotting analysis of p24 Gag or actin (as a loading control). dpi, days postinfection. f U87.CD4.CCR5 (left) or U87.CD4.CXCR4 cells (right) were transfected, infected, and analyzed by western blotting as in (e). p24 Gag intensity was quantified (day 2 and day 5), and represented as percentage relative to cells transfected with control siRNA (mean ± SD, n = 3). *p < 0.05. n.s. not significant, WB western blotting; dpi days postinfection