Figure 2. Involvement of death receptors in TSN-mediated apoptosis enhancement.

(A) A549 cells were treated with 200 nM TSN for indicated time. After treatment, RT-PCR analysis was performed to measure the expression of DR4 and DR5 at mRNA levels. GAPDH was included as a loading control; each experiment was performed at least in triplicate. (B) A549 cells were treated with TSN at increasing concentrations for 12 h. After treatment, mRNA levels of death receptors were assessed by RT-PCR analysis. GAPDH was included as a loading control; each experiment was performed at least in triplicate. (C,D) A549 cells were treated with 200 nM TSN for 12 h, cell surface expression levels of death receptor 4 (C) or death receptor 5 (D) were measured by flow cytometry. Each experiment was performed at least in triplicate. (E) A549 cells were transfected with siRNAs against DR4, DR5, or both for 24 h before drug treatments. After 24 h drug treatment, cells were collected and apoptosis was measured. Data represent the mean ± SEM from three independent experiments, ***P < 0.001, as compared with cells transfected with siRNA control. (F) A549 cells were pretreated with Act D at 5 ug/ml for 30 min, and then subjected to 200 nM TSN treatment for 12 h. RT-PCR analysis was performed to assess death receptors 4/5 expression. GAPDH was included as a loading control; each experiment was performed at least in triplicate.