Elevated Axl expression in KS cells resulted from vFLIP expression or hypomethylation in AXL promoter. (A) The 293T cells were transiently transfected with expression vectors for KSHV latency genes (vFLIP, Kaposin B, LANA, and vCyclin) and lytic genes (vGPCR and vIL6). Empty pcDNA3 vector was used as control. Expression of Axl was analyzed by quantitative RT-PCR 72 hours after transfection and normalized to β-actin expression. (B) HUVECs stably transfected with a vFLIP-ER^TAM retroviral vector (HUVEC/vFLIP-ER) or a control vector³⁵  (HUVEC/vector) were grown on a 6-well plate and induced by 4-OHT (100nM) for 48 hours. The cells were treated with PI3K inhibitor LY (LY294002, 15μM, 5 hours), ERK inhibitor U0126 (15μM, 5 hours), and NF-κB inhibitor Bay (Bay 11-7085, 10μM, 2 hours), or vehicle dimethyl sulfoxide (DMSO). Cells were lysed, and expression of Axl was analyzed by Western blot (top panel) or quantitative RT-PCR (bottom panel). The relative protein level was quantitated by ImageJ (National Institutes of Health, Bethesda, MD), normalized to β-actin, and is shown below the Western blot. (C) LTC (top panel) and KS-IMM (bottom panel) were treated with PI3K inhibitor LY (LY294002, 15μM, 5 hours), ERK inhibitor U0126 (15μM, 5 hours), and NF-κB inhibitor Bay (Bay 11-7085, 10μM, 2 hours), or combinations of 2 of these 3. The whole-cell lysates were then subjected to Western blot analysis. (D) (Top panel) AXL copy number in LTC, KS-SLK, KS-IMM, HT29, and A549 cells was determined by quantitative PCR, as described in “AXL copy number analysis with quantitative PCR.” GAS6 was used as a reference gene. (Bottom panel) Genomic DNA isolated from 5 cell lines along with HUVEC/vector were converted by bisulfate, and the AXL promoter region was subsequently amplified, followed by cloning and sequencing. Total number of methylated sites from 8 clones was shown.