Rn1734

Rabbit anti-CLDN1, mouse anti-CLDN4, rabbit anti-ZO-1, Alexa Fluor 488 anti-mouse, and Alexa Fluor 555 anti-rabbit antibodies, and H₂DCFDA were obtained from Thermo Fisher Scientific (San Diego, CA, USA). Goat anti-β-actin antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-phosphoserine (p-Ser) and anti-phosphothreonine (p-Thr) antibodies were from Sigma-Aldrich (Saint Louis, MO, USA). LY, NiSPY-3, Quest Fluo-8 AM, rabbit anti-nitrotyrosine, rabbit anti-NOS1, rabbit anti-NOS2, rabbit anti-NOS3, and rabbit anti-ubiquitine were from Biotium (Fremont, CA, USA), Goryo Kagaku (Hokkaido, Japan), AAT Bioquest (Sunnyvale, CA, USA), R&D Systems (Minneapolis, MN, USA), Cell Signaling Technology (Beverly, MA, USA), ProteinTech (Rosemont, IL, USA), GeneTex (Irvine, CA, USA), and Stressgen Biotechnologies Corporation (British Columbia, Canada), respectively. NOC12, SIN-1, and L-NAME were from Dojindo Laboratories (Kumamoto, Japan). AMG9810, olvanil, and RN1734 were from FUJIFILM Wako Pure Chemical Corporation (Tokyo, Japan). DAF-2DA and FeTPPs were from Cayman Chemical (Ann Arbor, MI, USA). All other reagents were of the highest grade of purity available.

Cells were seeded in 96-well plates (Het-1A cells; 5.0 × 10⁴/well, KYSE-270 cells; 2.5 × 10⁴/well) or 24-well plate (Het-1A cells; 5.0 × 10⁵/well, KYSE-270 cells; 3.0 × 10⁵/well) or 12-well plates (KYSE-270 cells; 5.0 × 10⁶/well) and incubated overnight. The cells were treated for 2 h in acidic culture conditions (pH 6.5 to pH 3.5) or with chenodeoxycholic acid (CDCA; 200 or 400 μmol/L) (Wako Chemical, Osaka, Japan). The cells were then washed and cultured in fresh pH 7.2 medium for an additional 6 h. HST (1, 10, and 100 μg/mL) or inhibitors [NS-398 (Wako Chemical), pyrrophenone (Merck & Co., Kenilworth, NJ, USA), FR180204 (Merck & Co.), RN-1734 (Wako Chemical), HC067047 (Wako Chemical), AMG9810 (Tocris Bioscience, Bristol, UK), A784168 (Tocris Bioscience), GSK1016790A (Sigma-Aldrich, St. Louis, MO, USA)] were added to both acidic and pH 7.2 culture medium for 8 h. Finally, the culture medium was collected to estimate PGE₂ concentrations using the PGE₂ Enzyme Immunoassay Kit (Cayman Chemical Co., Ann Arbor, MI, USA).