Fig. 3

IKKε expression or activity suppresses non-canonical NF-κB protein expression. a) 50 μg of protein was analyzed in whole cell lysates of breast cancer cells engineered to express a constitutively active shRNA against the IKBKE transcript (shIKKε), left, or an expression vector for constitutive synthesis of IKBKE, right. Representative blots show efficiency of shIKKε and expression vector after 8 days selection in puromycin or neomycin, respectively. b) Left, using the MDA-MB-468 cells with stable IKBKE shRNA activity we measured the viability of the cells over a 9 day period. Loss of IKKε significantly impaired survival compared to control. This difference was abrogated in the presence of 1 μM MEK inhibitor. Right, the same experiment was performed using the BT549 cells stably transfected with the IKBKE expression vector. * day 9 significantly different from corresponding vehicle control P < 0.05, one-way ANOVA, post hoc Tukey. c) Left, western blot analysis of MDA MB 468 cells with shRNA-mediated knockdown of IKBKE shows decline in activated MEK (phosphorylated ERK1/2) and increase in p52 levels compared to negative control. Right, stable expression of wild type IKKε is correlated with increased activated MEK and decreased p52. Twenty-four hour exposure 2 μM IKKβ inhibitor had no effect on protein levels