Figure 3. Effect of vCyclin deletion on KSHV-mediated cell proliferation, (A) Deletion of vCyclin does not affect KSHV immortalization of primary cells. Cells were passaged every 3 days, and the cumulative total cell numbers were calculated. Mock cells underwent crisis at around passage 23, while WT, mutant, and revertant cells continued to grow without encountering any crisis. (B) vCyclin is required to maintain accelerated cell proliferation at contact-inhibited condition. Cells were seeded at 2 × 10⁴ cells/well in 6-well plates and counted daily. (C) Deletion of vCyclin reduced cell saturation density. Cells were seeded at 2 × 10⁵ cells/well in 6-well and cultured with daily change of medium. Total numbers of cells were counted at day 14 post-seeding. (D) Mutant cells form fewer and smaller foci than WT and revertant cells do in culture. Cells were cultured as described in (C) and foci were examined at day 14 post-seeding. (E–G) Mutant cells form fewer and smaller colonies than WT and revertant cells do in softagar assay as illustrated by images of the colonies (E), the numbers of colonies (F) calculated based on the plates described in (E), and a graph showing the distribution of colony sizes (G) calculated based on the plates described in (E).