PKC412-induced apoptosis is preceded by the production of ROS and is JNK dependent. (A) Treatment with PKC412 (5 μM) induces ROS production in HMCLs measured by H2DCFDA (1 μg/mL) fluorescence (⊡ indicates NCI-H929; ▪, LP-1). Graph represents data from 2 individual experiments. (B) PKC412-induced (5 μM for 30 minutes) ROS production was inhibited by 2 hours of pretreatment with LNAC (4 mM) as measured by H2DCFDA (1 μg/mL) fluorescence (▪ indicates LP-1; D, DMSO; P, PKC412; L, LNAC) (*P = .0014 versus PKC412 treatment, Student t test). Graph represents data from 3 individual experiments. (C) PKC412-induced (3 hours of treatment) modulation of c-jun and phosphorylation c-jun in HMCLs with and without pretreatment with the JNK-specific inhibitor SP600125 as shown by Western blot (P indicates PKC412; S, SP600125; D, DMSO). Representative image from 1 of 3 individual experiments is shown. (D) PKC412-induced apoptosis (4 μM PKC412) after 72 hours is partially abrogated by 30 minutes of pretreatment with SP600125 (25 μM) as determined by annexin V–FITC/PI flow cytometry of NCI-H929 (⊡) and LP-1 (▪). (*P = .027 and .043, respectively, versus PKC412 treatment alone, Student t test). Graph represents data from 3 individual experiments). Error bars for all graphs indicate SEM.
