AW464 activates JNK, P38 and ERK1/2 in MCF7 cells in a dose and time dependent manner

1. AW464 activates JNK, P38 and ERK1/2 in MCF7 cells in a dose and time dependent manner Phospho JNK Total JNK Phospho p38 Total p38 Phospho ERK1/2 Total ERK1/2 a tubulin DMSO 0.5 1 3 5 10 mM 0 5 30 60 180 360 min AW 464, 3h 5 mM AW 464

2. ASK1 – K709R mutation and transfection • AW464 causes dissociation of Trx1 from ASK1 • AW464 activates P38 and JNK, MAP kinases known to be downstream of ASK1 • Is this directly via ASK1 activation following dissociation from AW464? • K709R – represents a catalytically inactive mutant in which Lys709 has been replaced by Arg • Once activated through Trx1 release, ASK1 autophosphorylates at Thr845 • Over express ASK K709R and see if we can prevent MAP kinase activation and cell death caused by AW464. HEK293 IB: ASK HCT116 IB: Phospho ASK1 (Thr 845) IB: ASK Control ASK K709R c wt K709R c wt K709R

3. V5 catalase catalase Catalase - Overexpression • AW464 causes transient increase in ROS • ROS can dissociate Trx1 from ASK1 • Catalase “mops up” ROS • Can overexpression of catalase prevent AW 464 induced Trx1 dissociation from ASK1or cell death? HCT116 HEK293 con cat cat mitcat con cat cat mitcat • Two different catalase constructs. • One normal human catalase gene in mammalian expression vector • The other is same catalase gene with mitochondrial targeting sequence and V5 epitope tag