Mek1 inhibitor pd98059

C57Bl6N females carrying mutation in one allele of Pax7 gene were crossed with 129Sv males and genotyped as described previously [33 (link)]. ESCs were generated from blastocysts recovered from F1(C57Bl6Nx129Sv) Pax7+/− females induced to superovulate by injection of 10 IU of pregnant mare’s serum gonadotropin (PMSG, Folligon, Intervet) followed 48–52 h later by 10 IU of human chorionic gonadotropin (hCG, Folligon, Intervet) injection, allowed to mate with Pax7+/− males of the same cross. Pregnant females were sacrificed 96 h after hCG injection. ESCs were derived and characterized as described previously [3 (link)], e.g., all the lines were genotyped and karyotyped. Medium for ESC derivation was composed of KnockOut Dulbecco’s modified Eagle’s medium (KnockOut DMEM, Gibco) supplemented with 10% serum replacement (SR, Gibco) with addition of nonessential amino acids (0.1 mM, Gibco), l-glutamine (2 mM, Gibco), β-mercaptoethanol (0.1 mM, Sigma-Aldrich), penicillin and streptomycin (5.000 units penicillin and 5 mg streptomycin/ml, Gibco), murine leukemia inhibitory factor (LIF, 1000 IU/ml, ESGRO, Chemicon International), and 12.5 μM MEK1 inhibitor—PD98059 (Sigma-Aldrich). All experiments were carried out on three wild type Pax7+/+ ESC lines (B3, B5, B8) and three knock-out Pax7−/− ESC lines (B4, AI7.15, T2M4), which we described previously [3 (link)].

Antibodies for biochemical studies were as follows from Cell Signaling Technology: anti-p-EGFR Tyr 1068 (catalog 2234), anti-p-Akt/PKB (catalog 9271), anti-Akt/PKB (total) (catalog 9272). Anti-p-ERK1/2 Tyr 204 (catalog sc7383), anti-ERK2 (total) (catalog sc154), anti-p53 (catalog sc126), anti-p21 (catalog sc397), anti-p27 (catalog sc528), anti-EGFR antibody for IP (A-10, catalog sc373746), anti-AR (catalog N20) was from Santa Cruz Biotechnology. Anti-cyclin D1 (catalog 33-3500) was from Invitrogen. Anti-EGFR for WB (catalog 06-847) was from Millipore. Anti-p-Ser15-p53 (catalog MAB1839) was from R&D Systems. Anti-ERβ (catalog 06-629) was from Upstate. Anti-tubulin was from Sigma-Aldrich. The tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR), ZD 1839, was from Selleckchem. Bisphenol A (BPA) was from Sigma-Aldrich. The antiandrogen bicalutamide (Casodex) was from Sigma-Aldrich. The antiestrogen ICI 182,780 was from Astra-Zeneca. The MEK-1 inhibitor PD 98,059 was from Sigma-Aldrich. All other reagents were of chemical grade.