MEK-dependent up-regulation of Bim-EL confers the DCC-2036 induced apoptosis.

<p>A, knockdown of Bim attenuated the apoptosis in EOL-1 cells. EOL-1 cells were transfected with human Bim siRNA or non-specific control pool (Mock siRNA), respectively. Then cells were exposed to the indicated concentrations of DCC-2036. Twenty-four hours after treatments, the levels of indicated proteins were analyzed with immunoblotting (<i>top</i>). Cell death was detected with trypan blue in triplicate (<i>bottom</i>). Columns, mean; error bars, SD. One-way ANOVA with <i>post hoc</i> intergroup comparison with control by Tukey test. ***<i>P</i><0.0001. B, DCC-2036 decelerated the turnover rate of Bim-EL protein. BaF3 cells expressing WT or T674I FIP1L1-PDGFRα were exposed to 400 nM DCC-2036 for 2 hours, and then cycloheximide (CHX) was added (100 μg/mL). Cells were harvested at the indicated time points and underwent the Western blotting. C, DCC-2036 elicited a proteosome-dependent degradation of Bim-EL protein. After a 2-hour pretreatment with MG132, BaF3-T674I cells were also treated with or without 400 nM DCC-2036 for 12 more hours. Then cells were harvested, and the levels of Bim-EL protein were analyzed by Western blotting. D, MEK-dependent upregulation in Bim-EL protein in DCC-2036 treated cells. EOL-1 cells were treated with a MEK inhibitor (U0126, 20 μM), a PI3K inhibitor (LY294002, 25 μM), DCC-2036 (6 nM) or combination of them, respectively, for 24 hours, then cells were harvested and the indicated proteins were analyzed by immunoblotting. E, DCC-2036 decreases Bim-EL polyubiquitination. BaF3-T674I cells were transfected with Bim-EL along with His-ubiquitin (His-Ub). After 24 hours, the cells were treated with or without DCC-2036 at 400 nM for 8 hours, and then collected for <i>in vivo</i> ubiquitination of Bim-EL. The ubiquitinated BimEL was quantified by densitometry using Image J. The scale of ubiquitinated BimEL were normalized to that of relevant total BimEL, and then normalized to control. The ratio was shown. Ni–nitrilotriacetic acid (NTA), nickel bead precipitate; WCE, whole-cell extracts.