Fig. 1

Kaposin B binds to c-Myc for regulating endothelial cell angiogenic activities. a Nuclear distribution pattern of KSHV Kaposin B protein in cells. Immunofluorescence staining for Kaposin B proteins. HMEC1 cells with stable Kaposin B (KapB) expression were fixed, and Kaposin B proteins were detected using anti-Flag mAb, followed by anti–mouse IgG secondary antibody conjugated with FITC (green). Cell nuclei were counterstained with Hoechst 33342, while actin filaments with Texas Red phalloidin (Alexa Fluor 568). b HMEC1 cells stably expressing Kaposin B were subjected to the Transwell cell-migration assay (n = 3). c Kaposin B increases cell motility in HUVEC. Primary HUVEC stably transduced with Kaposin B or the vector control by lentivirus were used for Transwell cell-migration assays (n = 3). d Kaposin B enhances microvascular formation of HUVEC in an in vitro MatriGel angiogenesis assay. Pictures were taken after 6 h of incubation (left), and tube length was then measured and compared (right). e Schematic representation of miR-221/-222 proximal promoter. Three E-boxes (E1 and E2/3, in red) were found. f Co-immunoprecipitation assays show Kaposin B and c-Myc form a protein complex. Cell lysates were prepared from HMEC1 cells stably expressing Kaposin B. Five micrograms of anti-FLAG (clone M2; left panel), anti-HA (right panel) or isotype IgG control were incubated with 500 μl of cell extracts and then analyzed by western blotting with indicated mAbs. g The interaction between c-Myc and Kaposin B was independent of promoter DNA. Co-immunoprecipitation assays were performed with or without DNase pre-treatment on cell lysates prepared from HMEC1 cells stably expressing Kaposin B. Anti-FLAG (clone M2) or isotype IgG control were incubated with cell extracts, and pull-down products were analyzed by western blotting with anti-HA mAb for c-Myc (upper panel) or anti-FLAG M2 mAb for Kaposin B (lower panel). h-i Knockdown of endogenous c-Myc levels in Kaposin B(+) HUVECs inhibits Kaposin B-induced cell migration (h, left) and microvasculature formation (i, right) (n = 3)