P glycoprotein p gp

Cells were lysed in RIPA lysis buffer with proteinase inhibitor cocktail (Boster, Wuhan, China) and total proteins were extracted. Proteins were quantified using the BCA protein assay (Pierce, Rockford, IL, U.S.A.). Forty micrograms of proteins were separated by 10% SDS/PAGE, transferred on to PVDF membranes (Millipore, Billerica, MA, U.S.A.) and immune blotted with the respective primary antibodies at 4°C overnight. After washing three times, the membranes were incubated with HRP-linked secondary antibody at room temperature for 1 h. Protein bands were signaled using a chemiluminescence (ECL) reagent (Beyotime Biotechnology). The following antibodies were used in the present study with the respective concentration: p-glycoprotein (P-gp) (1:1000; Cell Signaling Technology, Danvers, MA, U.S.A.), Ki67 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), cleaved caspase-3 (1:1000; Cell Signaling Technology), β-catenin (1:400; Santa Cruz Biotechnology), c-Myc (1:1000; Cell Signaling Technology), cyclinD1 (1:800; Santa Cruz Biotechnology), β-actin (1:1000; Cell Signaling Technology), and IgG-horseradish peroxidase (HRP) (1:4000; Santa Cruz Biotechnology).

Fetal bovine serum (FBS), Dulbecco’s modified Eagle medium DMEM, and Dulbecco’s phosphate-buffered saline (DPBS) were obtained from Invitrogen (Carlsbad, CA, USA). Penicillin-streptomycin was purchased from GenDEPOT (Katy, TX, USA). Sodium butyrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide (MTT), and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) were supplied by Sigma-Aldrich (St. Louis, MO, USA). Antibodies against p21; cyclin A; cyclin D1; cyclin E; Bad; acetyl-CoA carboxylase (ACC); ATP citrate lyase (ACLY); fatty acid synthase (FASN); LC3B; Beclin-1; p62; phospho-AMPKα (Thr172); AMPKα; mammalian target of rapamycin (mTOR); phospho-mTOR (Ser2448); Akt; phospho-Akt (Ser473); phospho-ACC (Ser79); carnitine palmitoyltransferase 1A (CPT1); breast cancer resistance protein (BCRP); Bax; P-glycoprotein (P-gp); and poly ADP ribose polymerase (PARP) were purchased from Cell Signaling Biotechnology (Beverly, MA, USA). Actin, short-chain acyl-CoA dehydrogenase (SCAD), and p53 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Long-chain acyl-CoA dehydrogenase (LCAD) antibody, propidium iodide, and RNase A were purchased from Abcam (Cambridge, UK). All other chemicals and reagents were of analytical or HPLC grade and were used without further purification.