Bml 257

The human epidermoid carcinoma A431 cell line, which overexpresses CCKBR, was generated and kindly provided by Dr. Luigi Aloj 23 (link). The A431 and A431/CCKBR cell lines were cultured in DMEM, whereas the rat pancreatic acinar AR42J cells (ECACC, UK) were grown in RPMI medium, supplemented with 10% FCS, 2 mM glutamine and antibiotics (0.1 mg/mL streptomycin, 100 IU penicillin) at 37 °C and 5% CO₂. For CCKBR-specific knock-down, duplex siRNAs against CCKBR or control duplex against luciferase (Microsynth) were used at a final concentration of 100 nM in Optimem (Gibco): CCKBRseq1 sense RNA 5′-UAUACGAGUAGUAGCACCAdTdT-3′, CCKBRseq2 sense RNA 5′-CCGCCAAAGGAUGGAGUACdTdT-3′ and control sense RNA 5′-CGUACGCGGAAUACUUCGAdTdT-3′. Cells at 60-80% confluence were transfected with siRNAs by using Lipofectamin 3000 according to the manufacturer's recommendations. RAD001 (Selleckchem), rapamycin (Enzo Life Sciences), BML-257(Enzo), SC-514 (Enzo) or metformin (Selleckchem) were diluted in DMSO or water, respectively.

Phorbol 12-myristate 13-acetate (PMA), N-acetylcysteine (NAC), dibenziodolium chloride (DPI), trolox, L-ascorbic acid, and cytochalasin D were purchased from Sigma-Aldrich (St. Louis, MO, USA). MitoTEMPO, Go6976, PD98059, and BML-257 were purchased from Enzo Life Sciences (Farmingdale, NY, USA). BB-Cl-amidine, and 4-aminobenzoic acid hydrazide (ABAH) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Chloroquine was purchased from Invivogen (San Diego, CA, USA). GW311616A was purchased from Adipogen Life Sciences (San Diego, CA, USA). 1,2-Bis (o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl ester) (BAPTA-AM) was purchased from Thermo Fisher Scientific (Waltham, MA, USA).