Transfection of high-dose 3p-RNA induces robust apoptosis.

<p>(A) CAL27 cells were transfected with the indicated dose of 3p-RNA and BE-RNA at corresponding doses as the control for 16 h and then stained with Annexin V and propidium iodide. Apoptotic cells (Annexin V positive cells) are indicated as the percentage of gated cells. (B) Lysates of CAL27 or SCC4 cells transfected with indicated doses of 3p-RNA or control for 16 h were immunoblotted with a PARP antibody. (C) Lysates of CAL27 or SCC4 cells transfected with 5 µg/ml of 3p-RNA or control for the indicated times were immunoblotted with a caspase-3 antibody. (D) Lysates of CAL27 cells transfected with 5 µg/ml of 3p-RNA or control for the indicated times were immunoblotted with a PARP antibody. (E) CAL27 cells transfected with 3p-RNA (5 µg/ml) or BE-RNA (5 µg/ml) for 4 h were stained with Rodamine 123 and propidium iodide. Apoptotic cells (R123 negative cells) are indicated as the percentage of gated cells. (F) CAL27 cells were pre-transfected with RIG-I-specific siRNA or negative control (si-Mock) 36 h before transfection with indicated doses of 3p-RNA for 16 h. Cell lysates were assayed by Western blot using a PARP antibody. β-actin was used as the loading control in (B), (C), (D), (F). Similar results were obtained in multiple repetitions (at least three) of these experiments.