Figure 2

Gain of function of RhoA caused by simvastatin through reducing HDAC1 and p190RhoGAP, and increasing p190RhoGEF expression in 3MC-treated cells. (a) hRECs were transfected with siHDAC1 overnight, followed by a 3-h 3MC challenge. The effect of HDAC knockdown in 3MC-mediated tumor-promoting effect was examined by Western blot analysis of the indicated molecules. (b,c) hRECs were pretreated with SAHA or simvastatin (SIM) for 1 h, followed by a 3-h 3MC challenge, and the resulting cell lysates or cytosolic, nuclear, or membrane fractions were analyzed by Western blots or RT-PCR, with GAPDH, lamin A/C, and VE-cadherin as input controls for the corresponding preparations. (d) hRECs were sequentially pretreated with either DMSO or ITSA (50 μM, an HDAC activator via TSA suppression) for 5.5 h, TSA (50 nM, an HDAC1 inhibitor) for 18.5 h or SIM (5 μM) for 1 h, followed by 3MC treatment for 3 h. The resulting cells were harvested and analyzed for the indicated proteins by western blots. (e) hRECs and Caki-2 cells were also pretreated with simvastatin for 1 h prior to a 3-h 3MC challenge to compare the therapeutic effect of simvastatin in Caki-2 malignancy with its protection in normal hRECs. The resulting cell lysates were partitioned into cytosolic–membrane fractions to analyze the activated forms of RhoA and CD44 in the membrane. The bar chart and Table S2 show the normalized intensity of each protein band obtained using GAPDH in Western blots. Results are expressed as the mean ± SD (n = 3). Data from a representative experiment are shown. Significant difference: *P < 0.05 and **P < 0.01 vs. control; ^#P < 0.05, and ^##P < 0.01 vs. 3MC treatment alone; ⁺P < 0.05 and ⁺⁺P < 0.01 vs. Caki-2 with scramble siRNA treatment; ^ψP < 0.05 vs. Caki-2 + DMSO. The gels have been run in the same experimental conditions and the cropped blots were shown. The entire gel pictures were shown in the Supplemental Fig. 2.