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. 2014 Mar 21;9(3):e92793. doi: 10.1371/journal.pone.0092793

Figure 3. Essential role of AhR/RhoA activation in pRb2/HDAC1 upregulation and nuclear translocation induced by 3MC in MCVECs.

Figure 3

Cells were transfected with siAhR (A), DNRhoA, or CARhoA (B) overnight, with or without 3MC treatment for 1 h and separated into membrane, cytosolic and nuclear fractions. Western blot analysis was used to examine Ras/c-Raf cytosolic-membrane distribution and AhR/pRb2/HDAC1 cytosolic-nuclear distribution by cellular fractionation. The effects of DNRhoA and CARhoA in cell proliferation were assessed by counting the cell numbers using a hemo-cytometer. Data are presented as mean ± SEM of 3 independent experiments. (C) Cells received a 1 h pretreatment with a ROCK inhibitor (Y27632) or a LIMK inhibitor (BMS-5), followed by 1 h of the 3MC challenge; the cells were then harvested and partitioned into nuclear and cytosolic fractions. Phosphorylation of myosin light chain and cofilin were used as positive controls for the actions of Y27632 and BMS-5, respectively. GAPDH, Lamin A/C, and VE-cadherin were used as internal controls for the cytosol, nuclear and membrane fractions, respectively, to verify equivalent loading. Representative results of 3 separate experiments are shown, and data are presented as the mean ± SEM (*P<0.05 vs. control or DMSO group; # P<0.05 vs. 3MC treatment alone).