I3C and DIM increase the expression of caspase-2 and decrease Bcl-2 both in vitro and in vivo.

<p>Whole splenocytes from C57BL/6 mice were cultured and activated with 1μg/ml of SEB, in the absence or presence of I3C or DIM (100µM) for 12 hours. qRT-PCR was performed on RNA isolated from these <i>in vitro</i> cultures to determine expression of caspase-2 (A) and Bcl-2 (C). RNA was also isolated from <i>in vivo</i> isolated mononuclear cells collected from SEB-induced acute liver injury mice after 24 hours to determine expression of caspase-2 (B) and Bcl-2 (D). Protein expression of both caspase-2 and Bcl-2 was evaluated by western blotting in both <i>in vitro</i> (E) and <i>in vivo</i> (F) experiments. Relative expression of activated caspase-2 (p19) and Bcl-2 are shown below each respective blot under the appropriate bands. Numbers for relative expression were calculated by first normalizing expression levels to β-actin and comparing them to the vehicle group using ImageJ software. Statistical significance (p-value <0.05) was determined using GraphPad Prism analysis software with one-way ANOVA and Tukey’s multiple comparison test (* indicates significance compared to SEB+Vehicle).