CDDO enhances granulocytic differentiation through CEBPA translation, and this involves the uORF of CEBPA. (A) HL60 cells were cultured for 22 hours with 0.01% DMSO or 0.5 μM CDDO after a 2-hour preincubation period with 20 μg/mL cycloheximide (CHX) and analyzed for CEBPA and beta-actin protein expression using Western blotting. (B) HEK293A cells were transiently transfected with an expression plasmid harboring the human CEBPA coding sequence and either a wild-type uORF sequence (uORFwt) or an uORF with an optimized Kozak sequence around the uORF ATG (uORFopt). Two hours after transfection, the cells were treated with 0.01% DMSO or 0.1 μM CDDO, and 24 hours after transfection, cells were lysed and subjected to Western blotting, using antibodies against CEBPA or beta-actin (left panel). (C) The ratio of p42/p30 was analyzed by densitometric analysis of panel B. (D) 32Dcl3, 6.15 + MigR1, and 6.15 + WT-uORF cells were cultured with G-CSF and treated with 0.01% DMSO or 0.5 μM CDDO for 48 hours, and Western blotting was performed using anti-HA, anti-HSP90, or anti–hnRNP-E2 antibodies. (E) May-Grünwald/Giemsa staining of 32Dcl3, 6.15 + MigR1, and 6.15 + WT-uORF cells cultured with G-CSF for 9 days in the presence or absence of 0.5 μM CDDO. (F) Western blot analysis of HL60 cells treated with DMSO or 0.5 μM CDDO. The blot was probed with CEBPA and hnRNP-E2 antibodies. HSP90 served as a loading control.