Figure 1. Lentiviral shRNA suppression of cyclin D1 and D2 induces cell-cycle arrest and delayed apoptosis in H929 tumor cells.

(A) Immunoblot analysis of cyclin D1, D2, and D3 expression in H929 myeloma cells 65 hours after infection with lentiviruses expressing cyclin D1 or cyclin D2 shRNA, NT control shRNA, or GFP cDNA. α-Tubulin levels are shown as loading controls. (B) Cell-cycle profiles of H929 cultures 4 days after lentivirus infection, comparing the effects of suppression of cyclin D1 or D2 with control NT lentivirus infection and with lentivirus-induced suppression of both cyclin D1 and cyclin D2. To ensure equivalent exposure to lentivirus and shRNA, cells infected with both D1 lentivirus and D2 lentivirus (D1 + D2) are compared with cells infected with D1 lentivirus and NT lentivirus (D1 + NT), D2 lentivirus and NT lentivirus (D2 + NT), or 2 aliquots of NT lentivirus (NT + NT). (C) H929 apoptosis was examined serially by flow cytometry (in this example on day 11) after cyclin D suppression. Viable cells (left lower quadrants) are distinguished from early apoptotic (annexin V–positive) or late apoptotic (propidium iodide–positive) cells. (D) Time course of H929 viability after infection with D1 + NT, D2 + NT, D1 + D2, or control NT + NT lentiviruses. Viability was determined by annexin V and propidium iodide staining, as illustrated in C, and is normalized to uninfected H929; 5% error in viability is shown.