(A, B) Western blot analyses of regulation induced by silencing RhoA or RhoC. (A) The regulation of NAG-1, p21Cip1, and SPARC expression was analyzed at the protein level. PC-3 cells were transfected with 20 nM of the indicated control and specific siRNA. At 48 h posttransfection, cells were lysed and analyzed by immunoblotting with specific antibodies to NAG-1, p21Cip1, SPARC, RhoA, RhoC, and Erk1/2. (B) The induction of NAG-1 was also observed in LnCaP and MCF-7 cells upon RhoC silencing by either siRhoC or siRhoC#2, again demonstrating that these regulations are not cell specific. At 48 h posttransfection, cells were lysed and analyzed by immunoblotting with specific antibodies to NAG-1, RhoC, and Erk1/2. (C, D) NAG-1 is involved in the regulation of anchorage-independent growth following RhoC silencing. (C) PC-3 cells or (D) LnCaP cells were transfected with 20 nM of the indicated control and specific siRNA. At 24 h after transfection cells were plated in soft agar as described in Materials and Methods. Results are reported as percentages of values obtained with the ctrC and are mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p< 0.001, ANOVA, followed by Tukey–Kramer analysis.