(A) RT-PCR analysis of RASSF1A and RASSF1C expression in breast cancer cell lines (MDA-MB231 and T47D) using RASSF1A and RASSF1C specific primers. The RT-PCR results clearly show that RASSF1C mRNA is expressed in both cell lines. Unlike RASSF1C, RASSF1A mRNA expression was not readily detected in MDA-MB23 and T47D cells. RASSF1A and RASSF1C cDNAs were used as templates as control for RASSF1A (1A-cont) and RASSF1C (1C-cont) primers and actin was used as an internal control. (B) MDA-MB231 and T47D breast cancer cells were transfected with psilencer-RASSF1C-258 (siRNA-1C) to silence RASSF1C and pSilencer (control) plasmids as previously described [18, 19]. The proliferation of cells treated with siRNA-1C was significantly reduced (P < 0.05, t-test) compared to cells transfected with the empty pSilencer plasmid. (C) Transfected MDA-MB231 and T47D cells were also incubated with 3H-thymidine and 3H-thymidine incorporation was assayed as previously described . Proliferation of cells treated with siRNA-1C was significantly reduced (P < 0.05 vs vector control, t-test) compared to cells transfected with control plasmid. Values are mean ? SEM of 8 replicas; experiments were performed at least three independent times. (D) RT-PCR analysis of RASSF1C expression in transfected MDA-MB231 and T47D either transfected with psilencer-RASSF1C-258 (siRNA-1C) or pSilencer plasmid alone (control). The RT-PCR results clearly show that RASSF1C mRNA was significantly reduced in cells treated with siRNA-1C compared to control. The amplification of actin as an internal control is the same in both treated and control cells. (E) T47D breast cancer cells were infected with Mission® lentiviral-shRNA-control particles (lentiviral-control) and Mission® lentiviral-shRNA-RASSF1C (lentiviral-shRNA-1C) to silence RASSF1C. The proliferation of cells infected with lentiviral-shRNA-1C was significantly reduced (P < 0.001, t-test) compared to cells infected with lentiviral-shRNA-control.