Mibolerone mib

Authenticated human BC epithelial cell lines MCF-7, ZR-75-1, MDA-MB-231, SKBR3 and LNCap were purchased from Interlab Cell Line Collection, Italy. Cells were stored according to supplier's instructions and used within 4 months after frozen aliquots resuscitations (less than 30 passages). Every 4 months, cells were authenticated by short tandem repeat analysis (AmpFLSTR Profiler Plus PCR Amplification Kit, Applied Biosystems) at our Sequencing Core; morphology, doubling times, estrogen sensitivity, and mycoplasma negativity (MycoAlert, Mycoplasma Detection kit, Lonza) were tested.
MCF-7 and ZR-75-1 were maintained in DMEM/Ham F-12 medium (1:1) (DMEM/F-12) supplemented with 5 % Fetal Bovine Serum (FBS). MDA-MB-231 were cultured in 10 % FBS DMEM, LNCaP were grown in RPMI 1640 supplemented with 10 % FBS. Additionally, culture media were supplemented with 100 IU/ml penicillin, 100 ng/ml streptomycin, and 0.2 mM L-glutamine. For experimental purposes, cells were synchronized in phenol red-free (PRF) and serum-free media (SFM) for 24 hours (h) and then switched to PRF-media containing 5% charcoal-treated FBS (PRF-CT) in the presence or absence of Mibolerone (Mib, from Perkin Elmer), 5α-Dihydrotestosterone (DHT, Sigma) and hydroxyflutamide (OH-Fl, from Sigma Aldrich). All media were purchased from Invitrogen, Italy.

LNCaP cells were cultured in 6‐well plates at a density of 4 × 10⁵ cells/well in hormone‐depleted medium, comprising phenol red‐free RPMI (Sigma) supplemented as above but with 5% charcoal‐stripped fetal bovine serum (Labtech International), for 72 h prior to treatment with 10 nM mibolerone (Mib) (PerkinElmer Life Sciences, Wellesley, MA), 1 μM bicalutamide (Bic) (Sigma, Chemical Co., St. Louis, MO) or 10 nM 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG) (Sigma). Cells were harvested for protein extraction and protein expression assessed by Western blotting using 10 μg of total lysate.