Effects of RhoA or Rock inhibition on MK polyploidization. (A) Effects of RhoA (TatC3) inhibitors on MK polyploidization. The ploidy level of CD41⁺/CD42⁺ MKs was analyzed at day 8 or 9 of culture (72 hours after addition of the inhibitors). The mean ploidy was calculated from the number of cells of each ploidy class in 3 independent experiments. Mean ploidy levels was 3.79N in the control and 5.69N with TatC3 (n = 3, P < .005). The ploidy histograms are illustrated on the right part of the figure. (B) Effects of Rock (Y27632) inhibitor on MK polyploidization. The mean ploidy levels was 3.67N in the control and 6.02N with Y27632 (n = 3, P < .001). (C) Effect of a RhoA dominant negative (RhoA N19) on MK polyploidization. The ploidy level of GFP⁺/CD41⁺/CD42⁺ cells was analyzed by flow cytometry at day 9 of culture. The mean ploidy was calculated from the number of cells in each ploidy class in 3 independent experiments. The mean ploidy level was 3.72N with RhoN19 and 3.2 N with the empty Migr (n = 3, P < .03). (D) The effect of Rock I shRNA was ascertained by Western blotting and real time RT-PCR. Western blot quantification showing that the protein levels of Rock I/Hsc70 (used as a control of protein loading) was reduced approximately 40% by the specific Rock I shRNA compared with the SCR shRNA (control). Real-time RT-PCR showing that the mRNA level of Rock I mRNA was reduced approximately 35% by the specific Rock I shRNA compared with SCR shRNA (control). (E) Effect of a Rock I shRNA on MK polyploidization. The mean ploidy level was analyzed as described above: SCR shRNA control (3.62N); Rock I shRNA (4.68N; n = 3, P < .04).