Fig. 6. Restitution of Runx2 protein levels during early G₁ phases of cell cycle is independent of transcriptional activity for mRNA synthesis.

(A) A specific experimental strategy using the RNA polymerase II inhibitor α-amanitin was developed to block the post-mitotic contribution of mRNA synthesis to Runx2 protein synthesis during G₁. MC3T3-E1 cells were synchronized by incubation for 16 h with nocodazole to generate a mitotic block. Mitotic cells were pre-treated with or without α-amanitin for 2 h and harvested by gentle agitation (0 h) and then released through mitosis into G₁ by washing and the addition of fresh culture medium supplemented with (+) or without (−) α-amanitin. (B) Cells were harvested at selected time points during the M/G₁ phase transition and interphase (0, 2, 4, 6, and 10 h). Progression through mitosis to G₁ phase in presence or absence of α-amanitin was monitored by flow cytometry. (C) Graphic representation of cell cycle stage data presented in panel B shown that α-amanitin treatment does not affect normal cell cycle progression of mitotic cells into G₁ phase. (D) Resumption of Runx2 protein levels at the M/G₁ transition in α-amanitin-treated cells or untreated control cells was analysed by western blotting. Cell cycle progression into G₁ was defined by monitoring cyclin D1 and Cdk4 protein levels. (E and F) Graphic representations of α-amanitin-treatment affecting post-mitotic change in Runx2 (no statistical differences between control and treated group), cyclin D1 (significant statistical differences between control and treated group from 6 h, P = 0.001) and Cdk4 (significant statistical differences between control and treated group from 2 h, P = 0.001). Protein levels normalized to actin.