Zhu et al.

Synergy between Kaposi's Sarcoma-associated Herpesvirus (KSHV) vIL-6 and HIV-1 Nef Protein in Promotion of Angiogenesis and Oncogenesis: Role of the AKT Signaling Pathway

Xiaofei Zhu, Yuanyuan Guo, Shuihong Yao, Qin Yan, Min Xue, Tingting Hao, Feng Zhou, Jianzhong Zhu, Di Qin and Chun Lu

Supplemental Figure Legends

Figure S1. Inhibition of AKT activity suppressed the enhanced effect of soluble Nef on vIL-6-induced cell proliferation. The lentivirus-mediated vIL-6-expressing HUVECs were incubated with soluble Nef for 48 h and further transfected with a dominant-negative AKT construct (AKT-DN) and a control plasmid, pSRα, for 48 h. The collected cells were seeded and a CCK-8 assay was performed for the cell proliferation at days 1-3 post-seeding. The results represent the mean ± SD; a representative experiment repeated six times is shown. *P < 0.05 and ***P < 0.001 for Student’s t-test. n.s., not significant for Student’s t-test.

Figure S2. Nef synergistically strengths vIL-6-induced angiogenesis via activation of AKT in nude mice.

(A). Expression of phosphorylated AKT was analyzed by Western blot analysis in Matrigel plugs induced by co-expressing vIL-6 and Nef endothelial cells. The relative level of phosphorylated AKT was determined by quantitative densitometry compared to total AKT. The relative value of phosphorylated AKT in Mock group was considered to be one for comparison.

(B). Inhibition of AKT activity inhibited the promoted effect of ectopic Nef on vIL-6-induced cell proliferation. vIL-6 and Nef co-expressing HUVECs were transfected with AKT-DN and the control plasmid, pSRα, for 48 h. The collected cells were seeded and a CCK-8 assay was performed for the cell proliferation at days 1-3 post-seeding. The results represent the mean ± SD; a representative experiment repeated six times is shown. **P 0.01 and ***P < 0.001 for Student’s t-test. n.s., not significant for Student’s t-test.

Figure S3. Nef promotes vIL-6-induced tumorigenesis in nude mice.

(A). Immunohistochemical staining analysis of the expression level of b-FGF and cyclin D1 (original magnification, ×200) in nude mice tumor tissues induced by vIL-6 plus Nef co-expressing NIH3T3 cells.

(B). Quantification of results in (A).

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