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. Author manuscript; available in PMC: 2011 May 1.
Published in final edited form as: Cancer Res. 2010 Oct 19;70(21):8526–8536. doi: 10.1158/0008-5472.CAN-10-1563

Figure 5. BRAFV600E regulates p21cip1 expression through MEK-ROS-JNK signaling towards endogenous FOXO4.

Figure 5

a) Scavenging of cellular ROS represses JNK activation, Thr223-FOXO4 phosphorylation and subsequent p21cip1 expression. Lysates of puromycin selected, untreated or NAC-treated (4mM, 24hrs) HEK293T cells were analyzed by immunoblotting. Cells were transfected and treated as in Fig. 3a and. b) Interference with MEK signaling represses JNK activation, Thr223-FOXO4 phosphorylation and subsequent p21cip1 expression. Experiment as in a), except with pretreatment for 24hrs with the MEK inhibitor U0126 (20μM). c) FOXO4-induced p21cip1 transcription in Colo829 cells requires MEK activity and cellular ROS. p21cip1-luciferase assay from lysates of Colo829 cells expressing HA-FOXO4 following 24hrs pretreatment with 10μM U0126 or 4mM NAC. d) Endogenous FOXOs mediate BRAFV600E-induced p21cip1 transcription. A14 cells expressing BRAFV600E, short hairpins against FOXO1+3a and FOXO4 or a scrambled sequence and a FOXO4-mutant, insensitive to its corresponding short hairpin (HA-FOXO4-SM) were subjected to a p21cip1-luciferase assay. High levels of BRAFV600E were transfected (2μg (++) compared to 200ng otherwise used throughout the study to force higher p21cip1 transcription.