Figure 5

Effect of TRPC6 activators and gene silencing of TRPC6 on growth of breast cancer epithelial cell lines. (A) MCF-10A, MCF-7 and MDA-MB-231 cell lines were seeded into six-well plates and incubated for 24 hours at 37°C. Subsequently, hyperforin was added at a concentration of 10 and 1 μM and the cells were incubated at 37°C. As controls, cells were also mock-treated with DMSO (which was used to dissolve the hyperforin). At 0, 24, 48 and 72 hours post incubation cell growth was measured using a MTT assay (black square 10 μM, grey square 1 μM hyperforin and empty square mock-treated). (B) MCF-10A, MCF-7 and MDA-MB-231 cell lines were seeded into six-well plates and incubated for 24 hours at 37°C. Subsequently, hyperforin was added at a concentration of 10 and 1 μM and the cells were incubated at 37°C. As controls, cells were also mock-treated with DMSO (which was used to dissolve the hyperforin). At 72 hours post incubation viability was measured and normalized against the mock-infected cells (black square 10 μM and grey square 1 μM hyperforin). (C) TRPC6 in MDA-MB-231 cells was silenced using a SiRNA double transfection method. At 72 hours post the second transfection the levels of TRPC6 was ascertained by protein extraction and Western blotting. As loading controls the lower part of the gel was removed and probed with an antibody to β-actin. Two SiRNAs were utilized and compared to a SiRNA control. (D) TRPC6 in MDA-MB-231 cells was silenced using siRNA transfection. At 72 hours post the second transfection cell growth was ascertained using a MTT assay. Two SiRNAs were utilized and compared to a SiRNA control.