B2r sirna

Cells were seeded in 6- or 24-well clusters or a T-25 culture flask and then incubated at 37°C in 5% CO₂ until 80% confluent. B2R siRNA was purchased from Santa Cruz, USA. siRNA transfection solution was prepared according to directions provided by Santa Cruz to make a siRNA concentration of 400 nM. The cells were washed once with siRNA transfection medium (Santa Cruz, USA). Then, the appropriate siRNA transfection medium and siRNA transfection solution was added to each well. The cells were then incubated for 6 hours at 37°C in 5% CO₂. The transfection mixture was then removed and replaced with normal growth medium and incubated for an additional 24 hours. The cells were then treated with either H₂O₂ alone or 0.1 nM BK plus H₂O₂ as described above. SA-Gal staining was then used to determine senescence and DCFH-DA probes visualized using a fluorescence microscope at an absorption wavelength of 488 nm and emission wavelength of 530 nm as described above.

Alpha-MEM, EGF, bFGF, propidium iodide (PI), and RNAase A were purchased from Invitrogen (Hong Kong, China). HOE140, 5-Bromo-2′-deoxyuridine (BrdU), and mouse monoclonal anti-BrdU antibody were from Sigma-Aldrich (St. Louis, MO USA). Rabbit polyclonal anti-Akt, anti-pAkt(Ser473), anti-ERK1/2 and anti-pERK1/2 antibodies were from Cell Signaling (Danvers, MA, USA). Mouse monoclonal anti-BDKRB2 antibody was from Millipore (Merck Millipore, Germany). B2R siRNA, mouse monoclonal anti-cyclin D1, mouse monoclonal anti-cyclin E, HRP-conjugated goat anti-rabbit or rabbit anti-mouse IgG antibody were products of Santa-Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Transwell permeable support polycarbonate membrane and other cell culture flasks and plates were purchased from Corning Inc. (NY, USA).