Influence of ST1346 and ATRA on the phosphorylation of MAP kinases and effect of MAP kinase inhibitors on the granulocytic differentiation of NB4 cells caused by ATRA and combinations of ATRA + ST1346.

(A,C,E) NB4 cells were treated for the indicated amount of time with vehicle, ATRA (0.1 μM), ST1346 (10 μM), or the combination of the 2 compounds. Total cellular extracts were subjected to Western blot analysis using polyclonal antibodies recognizing ERK, p38, or JNK and the corresponding phosphorylated forms, as indicated by the circled ‘p’. The positions of selected molecular weight markers are indicated on the right. The blots shown are representative of 5 independent experiments. (B,D,F) NB4 cells were treated for 3 days with vehicle (DMSO), ATRA (0.1μM), ST1346 (10 μM), the ERK inhibitor U0126 (10 μM), the p38 inhibitor PD169316 (10 μM), the JNK inhibitor SP600125 (10 μM), or the indicated combinations of the various compounds. Aliquots of the extracts were used for the determination of NBT-reducing activity. In the case of panel F, we also measured the level of JNK kinase activity on JNK immunoprecipitates, using c-Jun as a substrate. Equivalent amounts of immunoprecipitates were electrophoresed on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), blotted on nitrocellulose, and challenged with antibodies recognizing the phosphorylated form of c-JUN (serine/63) or JNK. The results are shown underneath the column graph. The number of NBT⁺ cells is also shown above each panel. Results are expressed as the means ± SD of 3 separate culture dishes and are representative of at least 3 independent experiments. **Significantly higher or lower than the corresponding group treated with ATRA + ST1346 (P < .01 according to the Studentt test).