Isochaihulactone induces NAG-1 expression via JNK1/2 activation, and isochaihulactone-induced cell death can be rescued by NAG-1 siRNA transfection. (A) Expression of EGR-1 and NAG-1 after treatment of LNCaP cells with 20 μM isochaihulactone for the indicated times. (B) NAG-1 expression of LNCaP cells pretreated with the p38 inhibitor SB203580 (20 μM), the JNK1/2 inhibitor SP600125 (20 μM), or the MEK1/2 inhibitor PD98059 (50 μM) for 1 h and then treated with 20 μM isochaihulactone for 24 h. (C) Suppression of isochaihulactone-induced NAG-1 expression in LNCaP cells by NAG-1 siRNA transfection. LNCaP cells were transfected with scramble siRNA (#) or 20 nM, 40 nM NAG-1 siRNA for 48 h using the RNAifect transfection reagent followed by treatment with 20 μM isochaihulactone for 48 or 72 h. Western blot analysis was performed for NAG-1. (D) Isochaihulactone-induced anti-proliferative activity was measured with the MTT assay in LNCaP cells transfected with scramble (#) or NAG-1 siRNA for 48 h and then treated with 20 μM isochaihulactone for 48 h. The data represent the means ± S.D. from three independent experiments. ***, P < 0.001 versus vehicle.