d.a.MKK6 expression leads to the modulation of expression levels of lineage-associated transcription factors. (A) HL60 cells transduced with d.a.MKK6 (MKK6) or control (CTRL) were induced with DOX (2 µg/mL). Cell morphology and monocytic marker (CD11b and CD14) expression of HL60 cells after 48 hours of d.a.MKK6 induction are shown (bar = 10 µm). Mean values ± SD were calculated from 3 independent experiments (**P < .01, ***P < .001). (B) d.a.MKK6 (MKK6) or control (CTRL) expressing HL60 cells were induced with DOX (2 µg/mL), and whole-cell extracts from an equal number of cells were analyzed at indicated time points. Blocking of the p38 signaling pathway by 10 µM SB203580 (p38 inh.) stabilized C/EBPα and inhibited c-Jun expression. JNK signaling pathway inhibition by 10 µM SP600125 (JNK inh.) had no effect. One representative blot of 3 experiments is shown. (C) Real-time RT-PCR assessment of C/EBPα and c-Jun expression in response to d.a.MKK6 expression. (D) Blocking proteasomal degradation by MG132 (10 µM) stabilizes C/EBPα. D.a.MKK6 was induced by DOX (2 µg/mL); after 6 hours, a proteasomal block was induced by adding MG132, and subsequently stabilization of C/EBPα levels was monitored for up to 4 hours (+1 to +4 hours). Stabilization of C/EBPα by MG132 interferes with c-Jun mRNA induction in d.a.MKK6-expressing HL60 cells (**P < .01). (E) c-Jun phosphorylation, assessed by western blotting, in response to d.a.MKK6 expression. Simultaneous d.a.MKK6 and d.n.c-Jun expression (defined as NGFR⁺GFP⁺) impairs the upregulation of CD14 and CD11b. Bars represent mean values ± SD of 3 independent experiments (*P < .05, **P < .005).