Figure 3. Sildenafil and chemotherapy-induced lethality is mediated through increased autophagy. (A and B) DAOY cells were transfected to express LC3-GFP-RFP. Twenty-four hours after transfection cells were treated with drugs and cells were examined 6 h and 12 h after treatment using a fluorescent microscope and the mean number of LC3-GFP⁺ and LC3-RFP⁺ vesicles determined in >40 cells (n = 3, ± SEM). *P < 0.05 greater than vehicle control. (C–F) DAOY, D283, and HOSS1 cells were transfected with scrambled siRNA (siSCR) or siRNA molecules to knock down expression of ATG5 or Beclin1 (siATG5, siBeclin1). Thirty-six hours after transfection cells were treated with drugs and viability determined by trypan blue exclusion (n = 3, ± SEM) ^#P < 0.05 less than corresponding value in siSCR control. (C upper) DAOY cells were treated with drugs as indicated. Cells were isolated 12 h after treatment and immunoblotting performed to examine the expression of the indicated proteins (n = 3).