CDK inhibition decreases Trib2 resulting in a block in AML cell proliferation. (A) PTX and flavopiridol dose response of U937 cells assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay at 24 hours. Data are expressed as percentage over untreated control. Values are expressed as mean ± SD (N = 2, performed in triplicate). (B) Cell cycle analysis of PTX, dibutyryl cAMP, and flavopiridol dose response in U937 cells at 24 hours. (C) Cell proliferation of PTX and flavopiridol dose response of U937 cells assessed by CFSE staining 96 hours after treatment. (D) Western blot analysis of protein lysates from U937 cells treated for 24 hours with different concentrations of (left) PTX, (center) dibutyryl cAMP, and (right) flavopiridol. Shown are Cdk6, Cdk2, pRB (phosphor-RB), E2F1, Trib2, and C/EBPα-p42. Actin, total RB, and histone deacetylase 1 (HDAC1) are shown as protein loading controls. (E) WT and Trib2^−/− (KO) total bone marrow cells were treated with different concentrations of (left) PTX, (center) dibutyryl cAMP, and (right) flavopiridol and assessed for apoptosis by AnnexinV/DAPI staining. (F) WT and Trib2^−/− (KO) HSCs were treated with different concentrations of (left) PTX and (right) flavopiridol and assessed for apoptosis by AnnexinV/DAPI staining. (G) WT and Trib2^−/− (KO) HSCs were treated with 2 mM PTX or 62.5 nM of flavopiridol (FLV) for 24 hours and plated in a CFC assay. Data presented are mean ± SD of duplicate cultures.