JNK signaling was activated by RASSF6 during the cellular response to cisplatin and radiation treatment.

<p>(<b>A</b>) S18 (left) and 5-8F (right) cells stably overexpressing RASSF6 (RF6) or transfected with an empty vector control (Vec) were treated with the indicated doses of cisplatin (upper panel, DDP, 6 µM for S18 and 8 µM for 5-8F cells) or radiation (lower panel, IR, 8 Gy for S18 and 5-8F cells). Protein was collected for Western blot analysis to evaluate the expression of RASSF6, phosphorylated JNK, total JNK, phosphorylated C-jun (ser63), phosphorylated C-jun (ser73), total C-jun, and GAPDH as a loading control. (<b>B</b>) S26 (left) and SUNE-1 (right) cells stably transfected with two RASSF6 shRNAs (KD1, KD3) or with a negative control sh-RNA (NC) were treated with the indicated doses of cisplatin (upper panel, DDP, 6 µM for S26 and 8 µM for SUNE-1 cells) or radiation (lower panel, IR, 8 Gy for S26 and SUNE-1 cells). Protein was collected for Western blot analysis of the expression of RASSF6, phosphorylated JNK, total JNK, phosphorylated C-jun (ser63), phosphorylated C-jun (ser73), total C-jun, and GAPDH as a loading control. (<b>C, D</b>) S18 cells stably overexpressing RASSF6 (RF6) or transfected with an empty vector control (Vec) were treated with cisplatin (DDP, 6 µM) or radiation (IR, 8 Gy) in the presence or absence (0.1% DMSO was used as a control) of the JNK inhibitor SP600125 (8 µM). The cells were then collected for (<b>C</b>) Western blot analysis of the expression of phosphorylated C-jun (ser63), phosphorylated C-jun (ser73), and total C-jun, with GAPDH as a loading control; and (<b>D</b>) flow cytometry analysis of apoptosis after exposure to cisplatin or radiation treatment, *<i>P</i><0.05, **<i>P</i><0.01, Student's t test.