17β estradiol e2

HepG2 cells were obtained from American Type Culture Collection—LGC Standards. Cells were maintained in DMEM high glucose (Life Technologies) with 10% FBS, supplemented with 1% mixture of antibiotics containing penicillin, streptomycin, and amphotericin B (Life Technologies, Monza, Italy). HepG2 cells were seeded at a density of 0.55 × 10⁶ cells/well in 6-well plates and incubated at 37 °C in the presence of 5% CO₂ for 24 h. The medium was then changed and replaced with DMEM without phenol red (Life Technologies) with 10% DCC-FBS, supplemented with 1% mixture of antibiotics containing penicillin, streptomycin, and amphotericin B (Life Technologies). Before treatment, medium was replaced with DMEM without phenol red with 1% DCC-FBS, supplemented with 1% mixture of antibiotics containing penicillin, streptomycin, and amphotericin B. Cells were treated with vehicle (ethanol) or 1 nM 17β-estradiol (E2) (Merck-Sigma) for 16 and 24 h.
Control and LERKO mice were given 50 μg/Kg 17β-estradiol (E2) or vehicle (VEH) by subcutaneous injection; 16 h after, adult hepatocytes were isolated and grown according to the protocol followed by Valverde et al. [36 (link)]. Prior to treatment, cells were incubated in serum and phenol red-free medium for 45 min at 37 °C; then cells were exposed to VEH or 1 nM E2, accordingly with the previous treatment in vivo, for 2 h, before gene expression analysis.

Estetrol (E4) was provided by Mithra Pharmaceuticals (Liège, Belgium). 17β-Estradiol (E2) was purchased from Merck (Milan, Italy). The GPER agonist G1 (1-[4-(-6-bromobenzol [1 (link), 3 (link)] diodo-5-yl)-3a,4,5,9b-tetrahidro3H5cyclopenta[c]quinolin-8yl]-ethanone) and antagonist G15 (3aS,4R,9bR)-4-(6-Bromo-1, 3-benzodioxol-5-yl)-3a,4,5,9b-3 H-cyclopenta[c]quinolone) were purchased from Tocris Bioscience (Merck, Milan, Italy). The MEK inhibitor trametinib was obtained from MedChemExpress (DBA, Milan, Italy). All compounds were dissolved in DMSO, except E2 and E4, which were solubilized in ethanol.

The following antibodies were used: anti-Epo receptor (kindly provided by Dr. Patrick Mayeux, Institut Cochin, Paris, France), anti-p44/42 ERK (phospho-ERK1 at Thr202 and Tyr204, and phospho-ERK2 at Thr185 and Tyr187), anti-ERK, anti-phospho-AKT (Ser473), anti-AKT, anti-phospho-STAT5 (Tyr694/699), anti-STAT5 (all from Cell Signaling Technology, Danvers, MA, USA), anti-β-actin (Merck, Darmstadt, Germany), and horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA). The following reagents were used: 17β-estradiol (E2, Merck, Germany), ICI 182,780 (Tocris Bioscience, Bristol, UK), Complete Mini EDTA-free (Roche, Basel, Switzerland), and Immobilon Crescendo Western HRP substrate (Merck, Germany).