The RCC cell line OS-RC-2, was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 medium, supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin (all from HyClone; GE Healthcare Life Sciences, Logan, UT, USA), in a humidified incubator at 37°C and 5% CO₂ (14). Following culture for 24 h, OS-RC-2 cells were treated with 1 µM Taxol (Shanghai Hualian Pharmaceutical Co., Ltd., Shanghai, China) for 0, 12, 24, 36 and 48 h. Alternatively, following culture for 24 h, the cells were pretreated with 10 µM MT depolymerizing agent Colchicine (Col; cat. no. C8190; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) or 2 µg/ml RhoA inhibitor C3 transferase (cat. no. CT03-A; Cytoskeleton, Inc., Denver, CO, USA) for 1 h, which was followed by treatment with 1 µM Taxol for 24 h 37°C and 5% CO₂. Untreated cells served as the control.

Cells were stained for MF, MT and RhoA as previously described (15,16). Coverslips were washed twice with phosphate-buffered saline (PBS) and examined under a confocal scanning microscope (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The pCI-neo empty vector were provided by Professor Ningsheng Liu, Nanjing Medical University (Nanjing, China). The control vector was produced by inserting the RhoA coding DNA sequence into the EcoRI-Sal I site of pCI-neo vector. The dominant-negative mutants vector RhoAT19N was constructed using Asp instead of Thr188 (mutations 849 A→T and 850 T→G) using the site-directed mutagenesis kit (cat. no. 200517; Agilent Technologies, Inc., Santa Clara, CA, USA). Cells were plated in 25-cm² culture flasks at a density of 5×10⁵ cells/flask. Following culture for 24 h, cells were transiently transfected with the control vector and RhoAT19N vector. Transfection was performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in accordance with the manufacturer's protocol. The concentration of the DNA plasmid used was determined by the size of culture dishes, also according to the manufacturer's instructions. Cells were analyzed at 40 h following transfection (17).

For flow cytometric analysis of the cell cycle, 1×10⁶ cells were harvested by centrifugation (room temperature, 184 × g, 5 min), washed with PBS and fixed with ice-cold 70% ethanol overnight. Fixed cells were treated with 25 µg/ml RNase A at 37°C for 30 min and stained with 50 µg/ml propidium iodide (Sigma Aldrich; Merck Millipore, Darmstadt, Germany) for 30 min in the dark. The fluorescence intensity of individual cells was measured using a flow cytometer (Beckman Coulter, Inc., Miami, FL, USA). At least 10,000 cells were counted using CXP Cytometer version 2.2 software (Beckman Coulter, Inc.) (18).

The expression of RhoA protein was measured by flow cytometry as previously described (6). Briefly, 1×10⁶ cells were fixed with 4% (w/v) paraformaldehyde for 30 min at 4°C, and permeabilized using 0.1% Triton X-100. Nonspecific antibody binding was blocked by incubation in 0.5% bovine serum albumin (BSA; Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at room temperature. Cells were subsequently incubated with primary RhoA antibodies (dilution, 1:500; cat. no. sc-418; Santa Cruz Biotechnology, Inc.) in 0.01 M PBS at 4°C overnight. Following this, the cells were incubated with a FITC-conjugated anti-mouse secondary antibody (dilution, 1:50; cat. no. BA1101; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 1 h at room temperature in the dark. The expression of RhoA was then measured by flow cytometry (BD Biosciences, San Jose, CA, USA).

The OS-RC-2 cells were lyzed in ice-cold radioimmunoprecipitation buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) containing PMSF protease inhibitor (cat. no. Amresco0754; Beijing Solarbio Science & Technology Co., Ltd.) and phosphatase inhibitors (cat. no. C500017; Sangon Biotech Co., Ltd., Shanghai, China). Equal quantities of protein (40 µg) were separated by 12% SDS-PAGE, prior to transferring proteins to polyvinylidene difluoride membranes (0.4 µm; EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% BSA in TBST (0.1% Tween-20) with 0.2% sodium azide and incubated with primary antibodies: RhoA (dilution, 1:500; cat. no. sc-418; Santa Cruz Biotechnology, Inc.), GTP-RhoA (dilution, 1:500; cat. no. 80601; Neweast Biosciences, King of Prussia, PA, USA) or GAPDH (dilution, 1:1,000; cat. no. 2118S; Cell Signaling Technology, Danvers, MA, USA) overnight at 4ºC. Following washing, membranes were incubated with a horseradish peroxidase-conjugated goat anti-mouse/rabbit IgG secondary antibody (dilution, 1:5000; cat. no. BA1050/BA1054; Wuhan Boster Biological Technology, Ltd.) at room temperature for 2 h, and chemiluminescence was performed using an Enhanced Chemiluminescence Detection kit (Shanghai Biyuntian Biological Co., Ltd., Shanghai, China), according to the manufacturer's protocol, with Tanon MP version 4.1.2 software (Tanon Science and Technology Co., Ltd., Shanghai, China) (19).

All experiments were repeated three times. Data are expressed as the mean ± standard deviation. One-way analysis of the variance and Fisher's least significant difference post hoc tests were performed to evaluate the differences among groups by SPSS v13.0 software (SPSS, Inc., Chicago, USA). P<0.05 was considered to indicate a statistically significant difference.

Taxol is an effective chemotherapeutic used to treat cancer patients; it binds to and stabilizes MT, causing abnormal MT aggregation (20). Immunofluorescent staining revealed that in the absence of Taxol, tubulin was widely distributed throughout the cytoplasm and accumulated in the perinuclear regions of cells. However, in cells treated with Taxol, a distinct rearrangement of MT was observed, including the formation of rigid MT bundles in the cytoplasm and the area surrounding the nucleus (Fig. 1A).

To determine the effect of Taxol-induced MT polymerization on the expression of GTP-RhoA and the cell cycle, western blotting and flow cytometric analysis were performed. Expression of GTP-RhoA was significantly upregulated following Taxol treatment in a time-dependent manner (Figs. 1B and C). Additionally, Taxol treatment caused an increase in GTP-RhoA protein expression in a dose-dependent manner (data not shown). The proportion of cells in G2/M phase increased >4-fold in cells treated with Taxol for 12 h, compared with the control (Fig. 1D). Therefore, these results suggested that Taxol-induced MT polymerization is accompanied by an increase in GTP-RhoA protein expression levels and cell cycle arrest.

To investigate the potential association between GTP-RhoA and MT arrangement, their cellular localization was determined via immunofluorescent staining. The results revealed a consistent co-localization of GTP-RhoA and MT structure (yellow colorization). GTP-RhoA and MT were widely distributed in control cells. The GTP-RhoA protein was located adjacent to the MT bundles in cells treated with Taxol. Following treatment with the MT depolymerizing agent (Col), the MT bundles were partially disrupted and the GTP-RhoA distribution dispersed. The results demonstrated a concurrent redistribution of MT arrangement and GTP-RhoA in Taxol treated cells (Fig. 2).

Next, the association between MT polymerization and GTP-RhoA protein expression was investigated. Combined treatment with Col and Taxol disrupted Taxol-induced MT bundling, however, little effect on MF arrangement was detected (Fig. 3A). The combination of Col and Taxol abolished Taxol-induced GTP-RhoA protein expression levels to the levels of the untreated control, as determined by western blotting (Fig. 3B) and flow cytometric analysis (Fig. 3C), and the Taxol-induced increase in the proportion of cells in G2/M phase was significantly reduced (Fig. 3D). These results suggested that MT arrangement regulated the protein expression levels of GTP-RhoA and cell cycle arrest.

To investigate the effect of GTP-RhoA on cytoskeleton arrangement and cell cycle, cells were pretreated with a RhoA inhibitor (C3 transferase) for 1 h or transfected with a RhoAT19N mutant to decrease GTP-RhoA expression (Fig. 4A). The inhibition of GTP-RhoA expression partially reduced the proportion of cells in G2/M compared with Taxol treatment alone (Fig. 4B and C). However, no marked effect on MT arrangement was observed in cells treated with the combination of C3 transferase or RhoAT19N mutant and Taxol (Fig. 4D). Thus, these findings confirmed that the cell cycle was affected by alterations in RhoA expression; however, GTP-RhoA expression did not influence MT arrangement.