Figure 3

Inhibition of ERK plus JNK sensitizes CEM-C1-15 cells to Dex-dependent apoptosis. Flow cytometric analysis of 20,000 collected events for each experiment. (A) Evaluation by Annexin-V staining and PI exclusion assay. CEM-C1-15 cells were pretreated for 24 hours with U0126 (U) plus SP600125 (SP) or cell permeable JNK inhibitory peptide (ip). Dex was then added and after a further 24 hours (for treatments with U+SP or U+SP+Dex) or 72 hours (Dex or U+ip or U+ip+Dex), cells were stained with Annexin-V-FITC/PI and examined by flow cytometry. Cells of sensitive clone CEM-C7-14 treated with Dex only are shown as a positive control. Abscissa: histogram of cells positive for Annexin-V; ordinate: cells positive for PI uptake. Note log scales, lower left quadrant shows viable cells; lower right, Annexin-V positive cells (early apoptosis); upper right, cell positive for both Annexin-V and PI (late apoptosis). Inset B is an assay for the block of ERK and JNK activity. Extracts of CEM-C1-15 cells treated with vehicle (C), ip, or SP were immunochemically tested for phosphorylation of c-Jun, n = 2. Extracts tested for ERK phosphorylation were treated with vehicle (C), Dex (D), U0126 plus SP600125 (USP), or the combination (USPD), n = 1.