Figure 1.

Reduction of BuGZ causes apoptosis in human cancer cells but senescence in primary foreskin fibroblasts (HFFs). (A) Depletion of BuGZ by siRNA treatment in the indicated cells. Two different amounts of control lysates (1/2 or 1) were loaded. Cells were analyzed 60 h after siRNAs transfection. α-tubulin, loading control. The numbers at the bottom of each lane indicate relative levels of BuGZ or Bub3 compared with controls, which were set to 1. Note that >90% of BuGZ was depleted by RNAi. (B) FACS assays for the quantification of apoptotic cells at 72 h after RNAi. Early apoptotic cells were Annexin V positive (see the bottom right quadrants), whereas late apoptotic ones were Annexin V and 7-aminoactinomycin D (7-AAD) double positive (the top right quadrants). All values are percentages of total cells, presented as mean ± SD from three independent experiments. (C) Representative microscopic fields of cells at 72 h after RNAi. Bar, 100 µm. (D) Growth curves of cells treated with control or BuGZ siRNA. Error bars indicate SD. Student’s t test: n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 from three independent experiments. (E) Stable knockdown of BuGZ with lentivirus-expressed shRNA in HFFs. The virus-infected cells were enriched by puromycin selection. The cell lysates were collected at day 5. (F) Growth curves of HFFs at the indicated days after shRNA transfection. Error bars indicate SD. Student’s t test: **, P < 0.01; ***, P < 0.001 from three independent experiments. (G) Increased senescence in BuGZ shRNA-transfected HFFs as judged by SA-β-gal staining (arrowheads). Bar, 50 µm. (H) Percentage of SA-β-gal–positive cells at the indicated days after shRNA transfection. Error bars indicate SEM. Student’s t test: ***, P < 0.001 from three independent experiments.