Figure 7. Effect of statins in preventing a 3MC-mediated decrease in cell-cycle regulatory proteins and in DNA incorporation induced by RhoA inactivation.

Cells were pretreated with simvastatin or pravastatin for 1-cytosol and nuclear-cytosol of 3MC-treated MCVECs was analyzed to determine the action of statins in RhoA inactivation and their effect in preventing 3MC-mediated alterations in c-Raf/pRb2/HDAC1/histone deacetylation. (A) We analyzed fractionation after 1 h; thereafter, the levels of cell-cycle regulatory proteins were assessed by Western blot after 4 or 6 h of 3MC treatment. (B) Cells were transfected overnight with a plasmid containing DNRhoA, and 3MC treatment was administered for 6 h. A western blot analysis was conducted to examine the effect of DNRhoA on the cell-cycle regulatory proteins reduced by 3MC. GAPDH (or α-tubulin), Lamin A/C, and VE-cadherin were used as internal controls for the cytosol (or total), nuclear and membrane fractions, respectively, to verify equivalent loading. (C) Cells were pretreated with statins for 1 h; this was followed by the 3MC challenge, which was pulsed for 15 h with BrdU (Invitrogen; 0.75 μg/mL) incubation for the DNA incorporation assay. Fixed cells on coverslips were stained with a mouse anti-BrdU antibody conjugated with Texas Red. Red represents BrdU-positive staining. Identical fields were stained with DAPI (Invitrogen) to reveal the positions of cell nuclei. We recorded micrographs of the representative fields at 200× magnification (scale bar in white  = 250 μm). (D) Cell numbers were counted using a hemo-cytometer at the indicated time points in cells with various treatments. Data are presented as mean ± SEM of 3 independent experiments (*P<0.05 vs. control group; ^# P<0.05 vs. 3MC treatment alone).