FIGURE 6.

Effects of NSC-741909 on MAP kinase phosphatase and JNK dephosphorylation. a and b, H460 cells were treated with 1 μm NSC-741909 for the indicated times (a) or treated for 1 h with the indicated concentrations of NSC-741909 (b). Levels of phospho-MKK7 (p-MKK7) (Ser-271/Thr-275) and MKP1 were detected by Western blot analysis. c, lysates from cells treated as in A were depleted of active JNK and then incubated with equal amount of active JNK at 37 °C for 30 min (top panel). The levels of remaining phospho-JNK (p-JNK) were detected by Western blot analysis. Active JNK added to a boiled cell lysate was used as a control for the initial amount of active JNK added (C1). The efficiency of JNK depletion in the cell lysates before adding active JNK was shown in the bottom panel. The cell lysate from H460 treated with 1 μm NSC-741909 for 4 h was used as a positive control (C2). d, H460 cells were treated with 1 μm NSC-741909 for the indicated times (top panel) or treated for 1 h with the indicated concentrations of NSC-741909 (bottom panel). Levels of MKP1 mRNA were detected by real-time PCR. The values were normalized with GAPDH mRNA levels. e, mock, control siRNA (Ctrl siRNA), or MKP1 siRNA-treated H460 cells were analyzed for cell apoptosis at 72 h after treatment. The values represent the means + S.D. of three analyses. **, p < 0.01, when compared with cells treated with control siRNA. f, Western blot analysis for MKP1 expression after knock-down by siRNA transfection.