Ly294002 akt

SNP and diethylamine (DEA) NONOate were purchased from Sigma Aldrich (MO, USA). The RBM3 antibody (ab134946) was from Abcam (Cambridge, MA, USA), and antibodies against, AMPKα (#5832), p-AMPKα (#2535), AKT (#9272), p-AKT (#4060), p-ERK1/2 (#4370), p38 (#9212), p-p38 (#4511), p-JNK1/2 (#4668), p-MKK3/MKK6 (#12280), cleaved PARP (#9541), and β-actin (#4970) were form Cell Signaling Technology (Beverly, MA, USA). Antibodies against ERK1 (sc-93), and JNK1 (sc-474) were from Santa Cruz Biotechnology (CA, USA). The 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Sangon (Shanghai, China). Annexin V-FITC cell apoptosis kit was from Beyotime (Jiangsu, China). Inhibitors SB203580 (p38 MAPK) and SP600125 (JNK), were from Sigma Aldrich. Inhibitors LY294002 (AKT) and U0126 (MEK) were from Cell Signaling Technology. The siRNAs specific to human Rbm3, human miR-143 mimics and antisense inhibitor, and their negative control oligonucleotides were purchased from GenePharma (Shanghai, China).

Exogenous BSA-AGE and control BSA were produced as previously published [17 (link)]. For examination of AGE treatment, cells (400,000) were seeded into each well of a 6-well plate in serum-free media overnight before treatment with AGE metabolite (50ug/ml). Effects of AGE treatment on the phosphorylation of ERα, AKT, and ERK was assessed by Western blot as indicated below. Pharmacological targeting of the MAPK/ERK and PI3K/AKT pathways in the presence of AGE was achieved in serum-free media using the molecular inhibitors U0126 (ERK) and LY294002 (AKT) (Cell signaling Technology, Danvers, MA) at a concentration of 10uM for 12 h before exposure to AGE metabolites. To examine the effects of AGE metabolite on tamoxifen resistance, cells (3,000) were seeded into each well of a 96-well plate and were treated with the active metabolite of tamoxifen, 4-hydroxytamoxifen (0, 10 µM) (Sigma-Aldrich, St. Louis, MO) in combination with varying doses of AGEs (0, 5 µg/mL, 10 µg/mL, 50 µg/mL). After 24, 48, and 72 h, SRB staining was used to quantify cell growth as described previously [18 (link)].