Arecoline induces detachment of HA22T/VGH cells, followed by apoptosis. (A) HA22T/VGH cells were treated with 0 (left), 30 (center), or 100 (right) μg/ml of arecoline for 24 h or 48 h, then cell morphology was observed under a phase-contrast microscopy at 200× magnification. The black arrows indicate detached cells. (B) After treatment of arecoline for 24 h, β1-integrin expression was measured by flow cytometric analysis using RPE-conjugated antibody. The histogram of red filled area is the untreated control and the black lines the treated groups. The values shown are the mean fluorescence intensity as a percentage of the untreated control value. (C) HA22T/VGH cells or primary normal rat hepatocytes were treated with the indicated concentration of arecoline for 72 h, then viable cells were counted using Trypan blue and the results expressed as a percentage of the untreated control value. (D) After treatment of arecoline for 72 h, the cells were harvested all together or the detached and adherent cells separately and genomic DNA extracted and analyzed on an agarose gel for DNA fragmentation. The left panel shows the total cells and the right panel adherent and detached cells separately. (E) TUNEL staining of the detached cells detected by flow cytometric analysis. The green filled area is the untreated control and the black lines the treated groups. The values shown are the percentage of TUNEL-positive cells in the detached cells. All data are the mean ± S.D. for three independent experiments. *: p < 0.05 as compared to the untreated control.