FIG. 3.
Loss of MnSOD alters cell morphology and increases proliferation in HepG2 cells. (A) Representative images of HepG2-sc (sc) and MnSOD-kd (kd) cells stained with crystal violet and DAPI. Images were taken using a 60×/1.00 oil immersion objective in bright-field to visualize crystal violet staining and fluorescence at an appropriate filter set for DAPI. (B) The effect of MnSOD deficiency on the proliferative ability was measured by counting of cells, BrdU assay, and MTT assay. Data are presented as fold induction (mean ± SD) normalized to HepG2-sc cells (*p < 0.05). (C) FACS analysis of apoptotic and necrotic cells after double staining with Annexin-V-FLOUS (AV) and PI at a sampling rate of 1 ml/min and wavelengths of λex = 488 nm, λem = 518 nm for AV and λex = 488 nm, λem = 617 nm for PI. Representative plot displaying AV versus PI shows three clearly separated populations of cells: R2 = live (AV neg, PI neg), R3 = early apoptotic (AV pos, PI neg), and R4 = late apoptotic/necrotic (AV pos, PI pos). (D) Alive (h), early apoptotic (ea), and late apoptotic/necrotic (a/n) cells analyzed by FACS. Data are mean ± SD of the fold induction normalized to HepG2-sc cells. BrdU, bromodeoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; PI, propidium iodide. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars