P21cip waf 1

Sodium hydrogen sulfide (NaHS), the anti-CSE antibody, PD98059 (an inhibitor of ERK), LY294002 (a PI3K inhibitor), 5-hydroxydecanoate (5-HD, a mK_ATP inhibitor), Chelerythrine chloride (Che, a PKC inhibitor) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The primary antibodies for anti-PKC-ε, anti-cleaved caspase-3 and -9, Bcl-2, cytochrome c (Cyt c), Na⁺/K⁺-ATPase, cyclin D1, p21^Cip/WAF-1 and GAPDH were from Santa Cruz (Bergheimer, Germany). Hoechst 33342, JC-1 kit and Calcein-AM were also from Santa Cruz. The anti-ERK1/2 and PI3K-Akt-GSK-3β antibodies were obtained from Cell Signaling Technology (Danvers, USA). Senescence β-galactosidase (β-gal) staining kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Rat advanced glycation end products (AGEs) ELISA kit was purchased from Proteintech Group (Wuhan, China). All other chemicals were from Sigma or Santa Cruz.

MCF-7 and MDA-MB-231 cells were grown up to 70–80% confluence and treated in 5% cs-FBS media before lysis in 500 µL of 50 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 2 mM sodium fluoride, 2 mM EDTA, 0.1% SDS, containing a mixture of protease inhibitors (aprotinin, phenylmethylsulfonyl fluoride, and sodium orthovanadate; Merck, Darmstadt, Germany) for total protein extraction. Equal amounts of proteins were resolved on 8% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane and probed with cyclin D1, p53, p21^Cip/WAF1 and GAPDH specific antibodies (Santa Cruz Biotechnology, CA, USA). The antigen-antibody complex was detected by incubation of the membranes with peroxidase-coupled goat anti-mouse or goat anti-rabbit antibodies and revealed using the ECL System (Amersham Pharmacia, Buckinghamshire, UK).