EGR-1 is the direct downstream target gene of JUN. (A) Western blot analysis compared OCI-My5 (OCI), OPM2, and U266 myeloma cells when untransfected (CON), carrying empty vector (EV), or overexpressing JUN (JUNH). Results showed specificity of JUN overexpression and effects on regulation of EGR-1. β-Actin was used as a loading control. (B) Effect of nonspecific scrambled shRNA (SCR) and JUN shRNA (JUN-KD) in U266 and OCI-My5 (OCI) cells on regulation of EGR-1 expression was evaluated by Western blot. Effects on cell viability in OCI-My5 (C) and OPM2 (D) cells for JUN overexpression (a), silencing of EGR-1 expression (b), JUN overexpression in the case of EGR-1 knockdown (c), overexpression of Survivin (d), JUN overexpression in the case of Survivin overexpression (e), and exposure to the pan-caspase inhibitor Z-VAD-fmk (f). Cell viability was evaluated daily by trypan blue exclusion. Error bars represent SEM for 2 independent experiments. U266 (E) and OCI-My5 (F) EGR-1–knockdown JUN overexpression cells compared with U266 and OCI-My5 JUN overexpression cell lines during exposure to 10nM bortezomib (Bort). Cell viability was determined every 12 hours by trypan blue exclusion. Error bars represent standard error of the mean for 2 independent experiments. (G) JUN binding to the proximal EGR-1 promoter in OCI-My5 and JUN-overexpressing OCI-My5 cells (OCI-My5-JUNH) was analyzed by ChIP by the use of antibodies against JUN or no antibody (negative control); real-time PCR followed.
