Both EBNA3C and Cyclin D1 are required for cell-cycle progression in EBV transformed cells.

(A) Lentivirus transduced short hairpin RNA vectors knock down EBNA3C and Cyclin D1 in EBV transformed LCLs. Transduction with sh-RNA-containing lentivirus and selection of EBV-infected cells (LCL1) with puromycin resulted in stable cell lines expressing specific si-RNA against EBNA3C (LCL1_sh-E3C), cyclin D1 (LCL1_sh-CyD1) and sh-RNA sequence that lacks any complementary sequences in the human genome (LCL1_sh-Cont). The selected cells with GFP fluorescence were monitored by fluorescent microscopy. (B) Western blots showing the expression levels of EBNA3C, pRb, Cyclin D1, Cyclin D2 and Cyclin D3 in LCLs. GAPDH was used as the loading control. (C) Approximately 1 million cells were plated into each well of the 6-well plates and cultured at 37°C in complete medium without puromycin. Viable cells from each well were counted by trypan blue exclusion method daily for twenty days using an automated cell counter. The results shown are representative of two independent experiments. Error bars show standard deviations.