Effects of CI-1040 in combination with ATRA on differentiation, histone posttranslational modifications, and expression of retinoid-responsive genes. (A) OCI-AML3 and (B) NB4 cells were pretreated with CI-1040 (0.5 μM) for 30 minutes and then exposed to ATRA (1 μM). CD11b membrane expression was then assessed by cytofluorimetric analysis after 48 hours. The red histogram refers to a matched IgG control; the black, green, and blue profiles refer to CD11b expression in vehicle control–, ATRA-, and CI-1040 + ATRA–treated cells, respectively. CD11b expression profile in CI-1040–treated cells was superimposable to vehicle control–treated cells and therefore omitted for clarity. One experiment representative of 3 performed with superimposable results is shown. (C) Histone H3 phosphorylation and acetylation status was assessed by Western blot analysis of nuclear extracts prepared after 24 hours of exposure to the indicated treatments, using phosphorylation- or acetylation-specific Abs (p-H3 and Ac-H3, respectively). Coomassie blue staining of the blotting membranes is shown as protein loading control (LC). One experiment representative of 3 performed with superimposable results is shown. (D) Relative RARβ transcript expression was assessed by quantitative real-time PCR, using specific primers, after 24 hours of exposure to the indicated treatments. Results are expressed as RARβ transcript fold induction relative to vehicle-treated control cells and represent the average ± SD of 3 independent experiments. (E) CD38 membrane expression was assessed by cytofluorimetric analysis in OCI-AML3 and NB4 cells, 48 hours after exposure to CI-1040 (0.5 μM) and ATRA (1 μM), either alone or in combination. Results are expressed as mean fluorescence index (MFI) and represent the average ± SD of 3 independent experiments.