Ag1478

The ovarian carcinoma cell line OVCA 433 was provided by Dr. Robert Bast Jr., M.D. Anderson Cancer Center, Houston TX [30 (link)] and grown in Minimum Essential Medium (MEME) (Gibco, Life Technologies, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA), 1 mM sodium pyruvate (Sigma, St. Louis, MO), 0.5 units/mL penicillin-0.5 μg/mL streptomycin (Gibco, Life Technologies), later referred to as MEME growth media. All Cells were maintained at 37°C under 5% CO2/95% air. Acute 10 μM cisplatin (Sigma-Aldrich) treatments were done in MEME growth media for 0–6 hours. EGFR inhibition was carried out by way of preincubation of the cells with 2 μM AG1478 (LC Laboratories, Woburn, MA) for 24 hours prior to cisplatin treatment. 10 nM EGF (Biomedical Technologies, Stoughton, MA) treatments for 15 minutes were performed as a positive control for EGFR activation.

The generation of psoriasis-like lesions in the ears of K5.Stat3C mice was conducted as previously described [14] (link), [21] (link). In brief, the skin lesions were generated by topical application of 0.68 nmol TPA (Wako) in 20 µl acetone to all surfaces of the left and right ears at day 0 and 2. Etanercept (Pfizer) (1 mg/mouse) was intravenously injected on day 0 and 2. The dosage of Etanercept was decided as previously described [22] (link). AG1478 (LC laboratories) was dissolved in acetone to prepare a 0.016% solution. Following this, 20 µl AG1478 solution was topically applied to all surfaces of the left and right ears twice a day. The concentration of AG1478 was decided as previously described [23] (link). Ear thickness was measured every day from day 0 to 3. The mice were sacrificed and ear skins were collected for gene and protein expression analysis and histological analysis. All mouse experiments were performed with strict adherence to institutional guidelines for minimizing distress.