Figure 2.

Knockdown of Bub1b inhibits RD and Rh30 cell growth and survival. To verify the shRNA library screening data, Rh30 and RD cells were infected with the Bub1b-specific clone shRNA3099, followed by 2 weeks of batch selection in medium containing puromycin. A, cells were divided into two aliquots and half were treated with doxycycline at indicated days, followed by protein lysate harvest. The Western blot demonstrates that both RD and Rh30 cells treated with doxycycline have markedly decreased Bub1b protein expression. B, cells released from double thymidine (2mM) synchrony were treated with or without doxycycline at indicated times and then harvested for Western blot analysis. C, cells were plated in 96 well plates at a density of 2000 cells per well and half were treated with doxycycline, followed by MTT measurement at days 3, 5, and 7. Data are mean ± SE (n =5). D, cells were plated in 6-well plates and half were treated with doxycycline for 12 days, followed by Neat Stain kit (astraldiagnostics) staining. The results are representative of 3 independent experiments. E and F, cells were infected with different Bub1b lentivirus shRNAs. After selection for10 days with puromycin, cells were read by MTT assay. Data are mean ± SE (n =4). G, Bub1b expression was examined in RD and SKOV3 cell lines after 6 days of selection.