Effects of manipulating CENP-A on cell proliferation and apoptosis in HepG2 cells.

HepG2 cells were transfected with CENP-A-expressing plasmids, siRNA-expressing plasmids, or empty vectors, as described in Materials and methods. The stable transfectants were subjected to the following experiments. A: Cell proliferation assay. The HepG2 transfectants were seeded in 96-well microplates in sextuplicate and cultured for up to 7 days. Cell proliferation was assessed by the MTT assay. **P#Pn = 3. B: Colony formation assay. The transfected or untreated HepG2 cells were plated in 6-well plates at a density of 1,000 cells per well. After 10 days, cells were stained with Giemsa and colonies consisting of >50 cells were scored. Representative dishes of three independent experiments are shown. C–E: Cell-cycle and apoptosis analysis. The transfected or untreated HepG2 cells were stained with PI alone or FITC-annexin V and PI and analyzed by flow cytometry. Representative histograms (C) and dot plots (D) of three independent experiments are shown. Apoptotic cells in D are FITC-annexin V positive and PI negative. E: Quantification of the cell-cycle and apoptosis analysis. The apoptotic index was determined as the ratio of apoptotic cell number to total cell number. #P##P*P** Pn = 3.