Fig. 4.

BCI-215 activates mitogen-activated and SAPK cascades in the absence of oxidative stress. (A) Activation kinetics. MDA-MB-231 human breast cancer cells were treated with BCI or BCI-215 (20 µM) for the indicated time points and analyzed for phosphorylation of the DUSP1/MKP-1 and DUSP6/MKP-3 substrates, ERK, JNK/SAPK, and p38, as well as their upstream activators MEK1 and MKK4/SEK1 by Western blot. (B) Activation of kinase cascades in three different cell lines. Cells were treated for 1 hour with vehicle (DMSO), 20 µM BCI-215 (215), or 5 µM doxorubicin (DOX). Data in (A) and (B) are from a single experiment that was repeated once. (C and D) ROS generation. MDA-MB-231 cells were prelabeled with Hoechst 33342 and chloromethyl-fluorescein diacetate, acetyl ester (CM-H₂-DCFDA) for 30 minutes followed by treatment with test agents for up to 5 hours. (C) At the indicated time points, cells were imaged and the percentage of ROS positive cells enumerated. (D) Concentration response at the 2-hour time point. Each data point is the mean of four wells ± S.E.M. from a single experiment that was repeated twice.