FIG. 5.

vIL-6 expression and VEGFR-1, -2, and -3 activation are not required for KSHV-induced lymphatic reprogramming. (A) TIME cells were infected with recombinant vIL-6-positive (vIL-6+) and vIL-6-negative (vIL-6−) KSHV for 48 h, and cell lysates were assayed by immunoblot analysis with the indicated antibodies. VEGFR-3 and podoplanin expression were induced by both recombinant viruses (lanes 2 and 3) but not in infected control BJAB cells (lane 1). (B) At 24 and 40 hpi, KSHV-infected TIME cells were treated with the indicated doses of the VEGFR Tyr kinase inhibitor KRN633, and cell lysates were harvested at 48 hpi for immunoblot analysis with the indicated antibodies. VEGFR-1, -2, and -3 activation is not required for the phosphorylation of Akt and STAT3 or KSHV-mediated lymphatic reprogramming. Lane M, mock-infected cells. (C) TIME cells were transfected with 3 μg of negative-control siRNA (lanes C) or the indicated VEGFR-1 or VEGFR-3 siRNA. After 24 h, cells were mock (M lanes) or KSHV (K lanes) infected and harvested at 48 hpi. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. VEGFR-3 siRNA did not block KSHV's induction of VEGFR-1 and podoplanin or the phosphorylation of STAT3 and Akt (left panels, lane 3). VEGFR-1 siRNAs did not block KSHV's induction of VEGFR-3 and podoplanin or the phosphorylation of STAT3 and Akt (right panels, lanes 3 to 5). *, cross-reactive band of the anti-podoplanin antibody.