Expression of Notch pathway genes in KS cells. (A) Expression of Notch receptors (top panel), Notch ligands (middle panel), and Notch pathway downstream target genes (bottom panel) in endothelial cells and KS cells was analyzed by RT-PCR. HUAECs, HUVECs, and LECs were included as controls. KSHV-infected LECs (LEC/KSHV) were treated with TPA (25 ng/mL) or dimethyl sulfoxide (DMSO) for 48 hours before harvest. KS-IMM was a tumor cell line isolated from KS lesion.²²  β-Actin expression indicates that an equal amount of RNA was used to perform PCR. (B) Expression of Notch3 in human KS tumor tissue was analyzed by immunostaining. The KS tumor regions are indicated by arrows. (C) Expression of Notch receptors and Notch ligands in KS cells were analyzed by Western blot. The relative expression level was quantitated by ImageJ (National Institutes of Health), normalized to β-actin, and is shown below each panel. (D) sDll4-Fc or human Fc fragment was clustered with anti-Fc antibody (1:1 ratio) at room temperature for 60 minutes. LEC/KSHV cells were then treated with clustered sDll4-Fc or Fc fragment (1 μg/mL) for 90 minutes, and whole cell lysate was subjected to Western blot with antibody against activated Notch.