FIG 11.

Effect of 5LO and LTB4 on fatty acid synthase gene expression. (A) FASN gene expression during primary infection of HMVEC-d. Cells grown to 80 to 90% confluence were serum starved for 8 h and infected with KSHV (30 DNA copies/cell) for different times. At different time points postinfection (up to 48 h), total RNA was isolated, DNase I treated, and then subjected to qRT-PCR using FASN-specific primers. (B) Expression of FASN in TIVE and TIVE-LTC cells. TIVE and TIVE-LTC cells were used to prepare total RNA, and the expression of FASN was analyzed using FASN-specific primers. (C, D) FASN expression in 5LO inhibitor-treated or 5LO-silenced and then KSHV-infected HMVEC-d (C) or 5LO inhibitor-treated or 5LO-silenced TIVE-LTC cells (D) was measured by the real-time PCR methods described in the text. Each bar represents the average ± SD of three independent experiments. (E) Lysates from sh-C- or Sh-5LO-transduced TIVE-LTC cells (a), BC-3 cells (b), or BCBL-1 cells (c) were Western blotted for 5LO and FASN, stripped, and immunoblotted for β-actin, and representative blots from three independent experiments are shown. (F) Effect of KSHV gene expression on FASN promoter activities. 293 cells were transfected with the control basic vector pGL3 or WT FASN promoter luciferase constructs. After 24 h, cells were serum starved, infected for different times, lysed, and assayed for luciferase activity as described previously. Data are expressed as the mean numbers of RLU after normalization with the cotransfected Renilla luciferase activity. Each reaction was done in triplicate, and each point represents the average ± SD of three independent experiments. (G) Effects of various concentrations of exogenous LTB4 on the FASN promoter. 293 cells transfected with WT FASN promoter luciferase constructs were serum starved (24 h) and stimulated with various concentrations of LTB4 for different times, lysed, and assayed for luciferase activity, as described in the text. Data are expressed as the mean numbers of RLU after normalization by cotransfected Renilla luciferase activity. Each reaction was done in triplicate, and each point represents the average ± SD of three independent experiments. (H) Effect of LTB4 in KSHV-infected culture supernatants on FASN promoter activity. Supernatants obtained from HMVEC-d treated with 5LO inhibitor or silenced for 5LO prior to KSHV infection were used to assess FASN promoter activation. 293 cells were transfected with control PGL3 basic and WT FASN luciferase constructs. After 24 h, cells were serum starved (10 h) and incubated with various supernatants obtained from uninfected or infected cells (as described above). Data are expressed as the mean numbers of RLU after normalization by the cotransfected Renilla luciferase activity. Each reaction was done in triplicate, and each point represents the average ± SD of three independent experiments. Percent inhibition was calculated by taking the level of FASN promoter activity in the presence of supernatant obtained from either untreated, solvent-treated, or sh-C-transduced cells to be 100%. The statistical analysis was carried out using a two-tailed Student's t test. *, P < 0.05; **, P < 0.01.