Cell culture

Alpha mouse liver 12 (AML-12) cells of male origin obtained from ATCC (Mannassas, VA), were seeded at 1×10⁴ per well in 12-well plates and grown to confluence for 24 hours in DMEM/F12 (GE Healthcare, Pittsburgh, PA) media supplemented with 10% fetal bovine serum, and 1% antimyotic/antibiotic solution. The cells were incubated in a 5% carbon dioxide atmosphere at 95% humidity and 37°C were sub-cultured every 2–3 days. Cells were treated for 30 minutes with either 20 ng/mL of epidermal growth factor (EGF) (Fisher Scientific, Pittsburgh, PA) as a positive control, 20 ng/mL of EGF and 20 μg/mL EGFR inhibitor (EI) (CAS 879127-07-8) (Millipore, Norwood, OH) as a negative control, or 20 ng/mL EGF and 20 μg/mL Aroclor 1260 (A1260). This Aroclor 1260 concentration was used based on previous work demonstrating that in vitro concentrations higher than 20 μg/ml were cytotoxic (Toxicity threshold = 26.7 ± 3.7 μg/mL in HepG2s) (Wahlang and others 2014a). The concentration of EGF was optimal to induce EGFR phosphorylation, and EGFR inhibitor was used at its IC₅₀ concentration for EGFR dephosphorization. A 30-minute treatment period produced robust EGFR phosphorylation but had no effect on PCB-mediated inhibition of EGFR phosphorylation. The positive and negative controls were used to define phosphorylated EGFR’s dynamic range. Media was removed by aspiration and the cells were washed with PBS. Cells were lysed in modified radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris, 150 mM NaCL, 1 mM EDTA/EGTA/β-glyceraphosphate/Na₃VO₄, and 1% Triton X-100) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO).

Human hepatoma-derived (HepG2) cells (male origin) obtained from American Type Culture Collection (ATCC, Manassas, MD) were plated 3×10⁵ cells per well in 12-well plates and grown to confluence in DMEM High Glucose media (GE Healthcare, Pittsburgh, PA) supplemented with 10% fetal bovine serum (FBS) and 1% antimycotic/antibiotic solution (Mediatech, Manassas, VA). HepG2 cells were treated and collected in a similar manner as the AML-12 cells. The Aroclor 1260 concentration-dependence assay was conducted in AML-12 and HepG2 cells similarly to aforementioned experiments with varied concentrations of Aroclor 1260 ranging from (0, 0.38, 0.94, 1.88, 5, 10, 15, 20 μg/mL) at constant EGF concentration. Aroclor 1254 was used at a concentration of 20 μg/mL similarly to the Aroclor 1260 experiments in HepG2 cells. The experiments with individual PCB congeners (PCB 3, PCB 6, PCB 8, PCB 9, PCB 138, PCB 149, PCB 151, PCB 153, PCB 170, PCB 174, PCB 180, and PCB 187 were performed with HepG2 cells at congener concentrations of 10 μM.