Notch signaling in KS cell survival and mural cell characteristic. (A) LEC, LEC/KSHV, and KS-IMM cells were treated by sDll4-Fc (4 μg/mL) or GSI-1 (1μM) for 48 hours. Purified human Fc fragment (4 μg/mL) and DMSO were used as control for sDll4-Fc and GSI-1, respectively. Cell viability was analyzed by MTT assay and normalized to control. (B) Cells were treated in the same way as in panel A but were harvested and subjected to quantitative RT-PCR. Expression of Notch pathway downstream genes was analyzed. (C) LEC/KSHV cells were transfected by Notch3 siRNA (20nM or 100nM) for 72 hours. Nonsilencing siRNA from QIAGEN (100 nM) was used as negative control. Cells were harvested, and Notch3 RNA level change after siRNA transfection was analyzed by quantitative RT-PCR. Expression level changes of putative Notch3 downstream genes were also analyzed. (D) Cells were treated in the way same as in panel C and subjected to Western blotting for Notch3 and α-SMA. The relative protein level of Notch3 and α-SMA was quantitated by ImageJ and normalized to β-actin level (E) Cells were treated as in panel C and subjected to MTT assay. Cell viability was normalized to mock transfection.