Sulindac sulfide

All animal procedures were performed in accordance with protocols approved by Institutional Animal Care and Use Committee of the University of California, San Francisco (IACUC protocol: AN187611) or the University of Pittsburgh (IACUC protocol: 13072053). Additionally, 5–6-week-old NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice were obtained from Jackson Laboratories (Bar Harbor, ME) or the UCSF Breeding Core Facility. Patient-derived xenografts (PDXs) were implanted into one flank of NSG mice under anesthesia and allowed to grow until palpable (~100 mm³). Mice (four mice with four tumors for each treatment group) were treated with vehicle control (saline), sulindac sulfide intraperitoneally (30 mg/kg; Sigma-Aldrich, St. Louis, MO, USA), erlotinib by oral gavage (50 mg/kg), or the combination of both as indicated throughout. Tumors were measured every other day by Vernier caliper in two dimensions and the volume was calculated as (larger measurement) × (smaller measurement)²/2.

Established type I human EOC R182 cells and 01-28 (MyD88-negative) primary EOC cells were kindly provided by Prof. Gil Mor (Yale University School of Medicine, CT, USA). A2780 type II human EOC cells and SKOV3 (MyD88-positive) cells were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were maintained in RPMI 1640 medium supplemented with 20% (v/v) heat-inactivated fetal bovine serum (FBS), 50 U/ml penicillin, and 50 mg/ml streptomycin (all from Welgene, Daegu, South Korea) in a 5% CO₂ humidified incubator at 37°C. Cell numbers were counted by trypan blue (Sigma-Aldrich, St. Louis, MO, USA) dye exclusion assay using a hemocytometer. Sulindac sulfide (≥98% HPLC) was purchased from Sigma-Aldrich. Recombinant NAG-1 protein was purchased from Peprotech (Rocky Hill, NJ, USA).

Cells were plated at 10,000 cells per well in 24-well plates and allowed to adhere overnight. The following day cells were treated in triplicate with vehicle (DMSO), erlotinib (Tarceva, Astellas Pharm Global Development Inc. Northbrook, IL USA), lapatinib (Sigma-Aldrich, St. Louis, MO, USA), sulindac sulfide (Sigma-Aldrich, St. Louis, MO, USA), or indomethacin (Sigma-Aldrich, St. Louis, MO, USA) at the indicated concentration of drug for 48 h. Cell growth inhibition was determined by MTT assay and normalized to vehicle-treated wells.

HCT15 cells (CCL-225; American Type Culture Collection) were treated with 50 µM sulindac sulfide (Sigma-Aldrich) for 1-24 h and analysed by western blotting following our previously established protocols (Mladenova et al., 2011 (link), 2013 (link)). Only adherent cells were analysed for protein expression. The membranes were incubated with primary antibodies for 1 h at room temperature or overnight at 4°C (β-actin, 1:10,000, clone AC15, Sigma-Aldrich), phosphorylated c-Jun (Ser73) no. 9164, c-Jun no. 9165 (Cell Signaling Technology), E-cadherin (1:2000, no. 610181, BD Biosciences), β-catenin (1:2000, no. 610153, BD Biosciences), p120-catenin (1:1000, clone H-90, no. sc-13957, Santa Cruz Biotechnology). ImageJ densitometry software (National Institutes of Health) was used for quantitative densitometry analysis.

Sulindac sulfide, aspirin, methanol, crystal violet, and G418 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against green fluorescent protein (GFP), Twist, Snail, vimentin, E-cadherin, α-tubulin, and GAPDH were obtained from Cell Signaling (Beverly, MA, USA). HMGA2 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).