CDK6 protein levels are down-regulated in c-Jun^Δ/Δp185^BCR-ABL–transformed cell lines. (A) Immunoblot analysis for CDK6, CDK4, CDK2, CYCLIN D2, CYCLIN D3, p16^INK4a, p15^INK4b, p21, and p27 protein expression in c-Jun^fl/fl- and c-Jun^Δ/Δp185^BCR-ABL–transformed cells. β-ACTIN served as the loading control. (B) Immunoblot analysis for c-JUN and CDK6 protein expression in c-Jun^fl/fl and c-Jun^Δ/Δ (top panel) and c-Jun^wt/wt and c-Jun^AA/AA (bottom panel) p185^BCR-ABL–transformed cells. β-ACTIN served as the loading control. (C) c-JUN and CDK6 protein levels of c-Jun^Δ/Δ and c-Jun^fl/fl cell lines after 2, 4, 6, and 8 weeks of transformation. β-ACTIN served as the loading control. One representative set of data is depicted. (D) [³H]-thymidine incorporation of the same cell lines was measured (n = 3; 2-tailed t test: c-Jun^fl/fl vs c-Jun^Δ/Δ 6 weeks, P = .002; c-Jun^fl/fl vs c-Jun^Δ/Δ 8 weeks, P = .0016). (E) Cdk6 mRNA levels of c-Jun^Δ/Δ and c-Jun^fl/fl cells 2, 4, 6, and 8 weeks after p185^BCR-ABL transformation were analyzed by quantitative PCR (n = 3; 2-tailed t test: c-Jun^fl/fl 2 weeks vs c-Jun^Δ/Δ 2 weeks, P = .0385; c-Jun^fl/fl 2 weeks vs c-Jun^fl/fl 8 weeks, P = .0293; c-Jun^Δ/Δ 2 weeks vs c-Jun^Δ/Δ 8 weeks, P = .0024). The fold change compared with c-Jun^Δ/Δ 2-week Cdk6 mRNA level is shown. Results were normalized by comparison with their Gapdh mRNA expression.