Insulin like growth factor igf 1

GC-1 cell line from American Type Culture Collection (ATCC, Manassas, VA, USA) was maintained in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 6% fetal bovine serum, FBS (Invitrogen). TCam-2 cell line (kindly provided by Prof. L. H. Looijenga, Erasmus MC-University Medical Center Rotterdam, Rotterdam, The Netherlands and P. Chieffi, University of Campania “Luigi Vanvitelli”, Caserta, Italy) was cultured in RPMI 1640 (Invitrogen) containing 10% fetal calf serum, FCS. Cell cultures were maintained in humidified 95% air and 5% CO₂ atmosphere at 37 °C. For treatments, 70% confluent cells were cultured in DMEM without phenol red and serum for one day and subsequently treated for 24 hours with 100 nM 17β-estradiol (E2), 10 nM 5α-dihydrotestosterone (DHT), 10 nM insulin-like growth factor (IGF-1) and 10 nM retinoic acid (RA) (Sigma-Aldrich Co., St. Louis, MO, USA).

The rat H9C2 cells were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (Gibco, USA), supplemented with 100 U/ml penicillin–streptomycin, and 10% fetal bovine serum (BSA) in a 5% CO₂ humidified atmosphere at 37°C. The cells were grown at a density of 4 × 10⁵ cells/ml. For the hypertrophy model, cells were grown with 10 μM insulin-like growth factor (IGF-1, Sigma-Aldrich, MO, USA) for 48 h at 37°C in a 5% CO₂ incubator.