Ethylisopropyl amiloride eipa

Tripterine was obtained from DESITE Biotechnology Co., Ltd (Nanjing, China). Soybean phosphatidylcholine (SPC, 95%) and 1, 2-stearoyl-3-trimethylammonium-propane (DOTAP) were provided by Avanti Polar Lipids, Inc. (Alabama, USA). Sodium deoxycholate (SDC), hyaluronic acid (HA), Hoechst 33258 and 3, 3’-dioctadecyloxacarbocyanine perchlorate (DiO) were purchased from Aladdin Reagents (Shanghai, China). Deionized water was prepared by a Milli-Q water purifier (Molsheim, France). Chlorpromazine, simvastatin, genistein, HUTCH-1 (monoclonal anti-CD44 antibody, rabbit), ethylisopropyl amiloride (EIPA) and Lantraculin A were purchased from Sigma-Aldrich (Shanghai, China). HPLC-grade methanol was supplied by Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All other chemicals were of analytical grade and used as received.

Cells seeded on glass bottom dishes were placed at 4 °C for 10 min and rinsed twice with ice-cold 0.01 M PBS + 2 mM CaCl₂/1 mM MgCl₂ at pH 7.4 (hereafter referred as PBS-2Ca). EZ-link Sulfo-NHS-SS-Biotin (100 mg powder, Thermo Scientific, MA, USA) is the sulfonated N-hydroxysulfosuccinimide-esters of biotin (NHS-B) used as surface protein biotinylation reagent, since it is not membrane permeable due to the presence of the charged sulfo group. We treated each dish with 1 mg NHS-B in 1 ml PBS-2Ca at pH 8.0 for 30 min at 4 °C. After rinsing with ice-cold PBS-2Ca (3 times per 3 min), we treated the cells with 200 µl of 10 µM streptavidin (Sigma Aldrich, MO, USA) in 1 ml PBS-2Ca at pH 7.4 for 30 min at 4 ℃. Following rinsing in PBS-2Ca, the cells were incubated with 1 ml of conjugated AuNP solution for 15 min at 4 ℃ followed by 15 min at 21 °C. Robust washing with 0.01 M PBS for five times was the final step before SERS or CFM analysis. During the spectroscopic analyses we added the isotonic buffer solution described in Conjugation of AuNP for SERS and CFM analyses. Isotonic buffer solution containing 100 µM ethyl-isopropyl amiloride (EIPA, Sigma Aldrich, MO, USA) was used to study the effect of the NHE inhibitor 30 min after addition. In case of CFM analysis the cells were also stained with nuclear dye Hoechst 33342 (Thermo Scientific, MA, USA).

ET‐1 (EMD Millipore) was used at 10⁻⁸ mol/L. The vehicle for ET‐1 was diH₂O. 10 μmol/L Y‐27632 (Cayman Chemical, Ann Arbor, MI) was used to inhibit ROCK. The vehicle for Y‐27632 was diH₂O. 10 μmol/L ethyl‐isopropyl amiloride (EIPA; Sigma) or 1 μmol/L dimethyl amiloride (DMA; Sigma) was used to inhibit NHE. The vehicle for both EIPA and DMA was dimethyl sulfoxide (DMSO).