Figure 3

Roscovitine inhibits DNA DSB-induced upregulation of cyclin A1 mRNA at doses primarily affecting CDK2 and post-translationally downregulates cyclin A1 protein levels over time in A549 cells. A) Expression levels respect to GAPDH (2^^-ΔCt), in mRNA of cyclin A1, cyclin A2, cyclin B, cyclin D and cyclin E after 24 hours of treatment with increasing doses of Roscovitine (5-60 μM). B) (Upper blot) Western blot analysis of inhibitory activity of Roscovitine (Rosc) against CKD2 phosphorylation of p130/Rb2 as shown by a shift in p130/Rb2 band height from hyper-phosphorylated in control cells to hypo-phosphorylated in Roscovitine treated cells, upper band is non-specific. (Lower blot) Western blot analysis of Roscovitine inhibition of CDK7 and CDK9 phosphorylation of the C-terminal domain (CTD) of RNA polymerase II, on serine 5 and serine 2 respectively, in cells treated for 24 hours with increasing doses of Roscovitine (10-40 μM). C) Fold change, respect to control (2^^-ΔΔCt), of cyclin A1 mRNA expression levels in cells treated with either increasing doses of Doxorubicin alone (250 nM to 5 μM) or increasing doses of Doxorubicin in combination with 20 μM Roscovitine for 24 hours. Note that black bars represent Doxorubicin only treated cells and correspond to the vertical axis on the left-hand side of the graph, while grey bars represent Doxorubicin and Roscovitine treated cells and correspond to the vertical axis on the right-hand side of the graph. D) Western blot analysis of cyclin A1, cyclin A2, CDK1 and CDK2 protein expression in cells treated for 24 hours with either Doxorubicin (750 nM or 2.5 μM) alone, 20 μM Roscovitine alone, or in combination (Dox 750 nM/2.5 μM + R). p53 protein expression was included as a control for drug treatments. E) Post-translational inhibition of cyclin A1 protein levels over time. (Left-side blot) cyclin A1 and p53 protein expression in cells treated for increasing amounts of time (6-72 hours) with 20 μM Roscovitine. (Right-side blot) cyclin A1 and p53 expression in cells treated for 24 hours with either Doxorubicin (750 nM and 2.5 μM) or 20 μM Roscovitine alone or in combination with 10 μM of the proteosome inhibitor MG-132.