rMC-1 (5 × 104) or HMCs (3 × 104) were treated as described, fixed in 4% paraformaldehyde, permeabilized with ice-cold acetone for 10 minutes, blocked with 1% BSA in PBS, incubated overnight at 4°C with antibodies against GAPDH (mouse anti-GAPDH for rMC-1, 1:800 dilution; rabbit anti-GAPDH for HMC, 1:200 dilution), and incubated in 5% goat serum, followed by 1-hour incubation with secondary antibody (anti–mouse secondary antibody conjugated to Texas red for rMC-1, 1:200 dilution; anti–rabbit antibody conjugated to Oregon green for HMC, 1:200 dilution) at room temperature. HMC cells were costained with an antibody to vimentin 1:200 dilution (secondary anti–mouse IgG conjugated to Texas red, 1:200 dilution). Coverslips were mounted on glass slides using antifade fluorescence mounting medium (Vectashield; Vector Laboratories, Burlingame, CA). GAPDH nuclear accumulation was detected with a fluorescence microscope (40× magnification; excitation, 540 nm; emission, 600 nm). Digital images were acquired (Image Pro-Plus; Media Cybernetics, Springfield, MD). rMC-1 was also analyzed using scanning laser confocal microscopy (LSM 410; Carl Zeiss Meditec, Göttingen, Germany) at 568-nm wavelength lines of an argon-krypton laser and an oil objective (100×, Plan-Neofluor; Carl Zeiss Meditec) The percentage of cells positive for nuclear GAPDH in four different fields per sample was determined, and the average values of several individual experiments were presented.