Molecular and chemical modulation of P^k expression. CD4⁺ HeLa cells (clone 1022) were either untreated (no vector) or transduced with control adenoviral vector alone (control [Ctrl] vector) or adenoviral vector containing full-length human P^k synthase (P^k-S) cDNA (P^kS vector). Both the control and P^k-S vectors contained an EYFP gene to detect transduction efficiency. After 48 hours, FACS analysis was performed and scatter plots of CD4⁺ HeLa cells labeled with anti–P^k GAM-FITC were analyzed, where background was compensated to isotype controls. (A) Histogram plots representing percentage of cell populations expressing EYFP (top panel) or P^k (lower panel) for no vector control (left), control vector (center), and P^k-S vector (right). (B) VT1 overlay for P^k detection was carried out on TLC of total GSLs extracted from control and transduced CD4⁺ HeLa cells. Lane 1: GSL standards. Lane 2: Cells without adenovector (no vector). Lane 3: Cells with control adenovector (control vector). Lane 4: Cells with adenovector-expressing P^k synthase gene (P^k-S vector). (C) HIV-1_IIIB (MOI, 0.1) was used to infect CD4⁺ HeLa cells with no vector, control vector, or P^k-S vector. After 3 days, HIV-1 infection was measured by p24^gag production. Percentage difference in HIV-1 infection was calculated based on CD4⁺ HeLa cells without adenovector (no vector). Data are representative of the mean plus or minus SEM where n = 3 infection data points; *P < .05 comparing P^k-S–transduced cells to untransduced cells. This figure is representative of 3 independent experiments. (D) Histogram plots representing percentage of cell populations expressing P^k after CD4⁺ HeLa cells (clone 6C) were either untreated (control) or treated with a GSL biosynthesis inhibitor (P4-treated, 2 μM) for 5 days to deplete glucosyl ceramide based GSLs, which includes P^k. (E) VT1 overlay for P^k detection was carried out on TLC of total GSLs extracted from untreated and P4-treated CD4⁺ HeLa cells. Lane 1: Control (untreated) cells. Lane 2: P4-treated cells. Lane 3-5: GSL standards. (F) HIV-1_IIIB (MOI, 0.1) infection of untreated or P4-treated CD4⁺ HeLa cells was measured by p24^gag production after 3 days of culture. Percentage difference in HIV-1 infection of P4-treated cells was calculated based on untreated control representing 100% infection. Data are representative of the mean plus or minus SEM where n = 3 infection data points; *P < .05. This figure is representative of 3 independent experiments