CDDO-induced CEBPA isoform changes require de novo protein synthesis, are associated with activation of eIF2α and eIF4E, but are independent of TGFβ and PPARgamma pathways. (A) HL60 cells were cultured for the indicated times in the presence of 1 μM CDDO with or without cycloheximide (20 μg/mL, CHX), and Western blotting was performed using the anti-CEBPA antibody. Blots were reprobed with anti–P-eIF2α, anti-eIF2α, anti–P-eIF4E, or anti-eIF4E antibodies. (B) Densitometric analysis of CEBPA p42/p30 and P-eIF2α/eIF2α bands from panel A. (C) HL60 cells were cultured for 24 hours in the presence or absence of 1 μM CDDO, and Western blotting was performed using anti-CEBPA, anti–P-eIF2α, and anti-eIF2α antibodies. In addition, cells were incubated in the presence of either the PPARgamma antagonist GW9662, calpain I inhibitor, an inhibitor of the eIF2α kinase PKR (2-aminopurine), or an inhibitor of the eIF2α phosphatase PP1/PP2A (calyculin A). (D) HL60 cells were treated with 0.01% DMSO, 0.1 μM CDDO, or 0.5 μM CDDO for 22 hours after a preincubation time of 2 hours with either no inhibitor or 1 μM of the TGFβ pathway inhibitor, SB505124, and analyzed for CEBPA and beta-actin by Western blotting. (E) HL60 cells were preincubated with the TGFβ pathway inhibitor (TGFβ/ALK5 receptor inhibitor) SB505124 for 2 hours and then stimulated with 10 ng/mL TGFβ1 for 1 hour and subjected to Western blotting using phospho-SMAD2 (P-SMAD2) or β-actin antibodies.