Fig. 2. Cyclopamine induces nSMase2, and downregulation of nSMase2 protects cells from drug-induced apoptosis.
(A) Effects of cyclopamine on nSMase2 (0, 6, 12, and 24 hours) were determined in Daoy cells with qPCR. Ribosomal RNA was used as a control. (B) roles of siRNA-mediated knockdown of nSMase2 versus nSMase1 in cyclopamine-induced caspase 3 activity (12 hours) were assessed. Nontargeting SCR siRNA-treated cells were used as controls. (C) effectiveness of siRNAs for targeting/knockdown of nSMase2 in the absence/presence of cyclopamine (10ug/mL) compared with SCR-siRNA-transfected controls was confirmed with qPCR. D and E, effects of siRNA-mediated knockdown of nSMase2 on cyclopamine-induced cell death were measured by AnnexinV-7-AAD staining (D), or ATP depletion (E) in Daoy cells. (F) Effects of down-regulation of nSMase2 on cyclopamine-mediated increase in N-SMase enzyme activity (n=2) were determined.