Figure 1. Co-treatment of KBV20C cancer cells with repositioned drugs DON or SID and HAL increased apoptosis. (A-B) KBV20C cells were plated on 96-well plates and grown to 30-40% confluence. The cells were then stimulated for 48 h with 50 ng/ml HAL (HAL), 5 μM donepezil (DON), 5 μM sildenafil citrate (SID), 50 ng/ml HAL plus 5 μM donepezil (HAL+DON), 50 ng/ml HAL plus 5 μM sildenafil citrate (HAL+SID), or 0.1% DMSO (Con). Cell viability assay was performed as described in “Materials and methods”. The data are presented as the mean±S.D. of at least two experiments repeated in triplicate experiments. Statistical analysis was conducted using one-way analysis of variance (ANOVA) followed by multiple-comparison test; *p<0.05 compared to the corresponding control. (C-D) KBV20C cells were grown on 60 mm-diameter dishes and treated with 50 ng/ml HAL (HAL), 5 μM donepezil (DON), 5 μM sildenafil citrate (SID), 50 ng/ml HAL plus 5 μM donepezil (HAL+DON), 50 ng/ml HAL plus 5 μM sildenafil citrate (HAL+SID), or 0.1% DMSO (Con). After 1 day, all cells were observed using an inverted microscope at ×10 magnification (scale bar=100 μm). (E) KBV20C cells were grown on 60 mm-diameter dishes and stimulated with 50 ng/ml HAL (HAL), 5 μM donepezil (DON), 5 μM sildenafil citrate (SID), 50 ng/ml HAL plus 5 μM donepezil (HAL+DON), 50 ng/ml HAL plus 5 μM sildenafil citrate (HAL+SID), or 0.1% DMSO (Con). After 24 h, Annexin V analyses were performed as described in Materials and Methods. (F) KBV20C cells were plated on 60 mm-diameter dishes and treated with 50 ng/ml HAL (HAL), 10 μM donepezil (DON), 10 μM sildenafil citrate (SID), 50 ng/ml HAL plus 10 μM donepezil (HAL+DON), 50 ng/ml HAL plus 10 μM sildenafil citrate (HAL+SID), or 0.1% DMSO (Con). After 24 h, western blot analysis was performed using antibodies against C-PARP, LC3B, Beclin1, Atg5, and β-actin.