Rn9893

hMSCs were cultured in complete medium containing inhibitors against stretch-activated ion channels 10 min prior to, and throughout, strain treatment, with vehicle only used as a control. Gadolinium chloride (GdCl₃; Sigma), dissolved in water and used at a final concentration of 10 µM (determined from a dose response experiment assessing its effects on nuclear morphology), was used as a broad-spectrum inhibitor against all stretch-activated ion channels^{27 (link)}. Amiloride (Sigma), dissolved in DMSO and used at a final concentration of 100 µM (determined from a dose response experiment assessing its effects on nuclear morphology), was used as an inhibitor against acid sensing ion channels (ASICs)^{27 (link)}. RN9893 (Sigma), dissolved in DMSO and used at a final concentration of 10 µM^{28 (link)}, was used to selectively inhibit the function of transient receptor potential vanilloid type 4 (TRPV4) ion channel. The tarantula venom peptide GsMTx4 (Abcam), dissolved in water and used at a final concentration of 3 µM²⁹ was used to inhibit piezo channels.

RN-9893 (Tocris Bioscience, Bristol, UK) was used as a TRPV4 antagonist. The efficacy of RN-9893 as a TRPV4 antagonist was previously validated (Wei et al., 2015 (link); Ahn et al., 2020 (link); Al-Shammari et al., 2020 (link)). RN-9893 was prepared by dissolving in 1% DMSO (Sigma-Aldrich, St. Louis, MO) and 1% Tween 80 (Sigma-Aldrich, St. Louis, MO, USA) followed by dilution in saline. The DMSO/Tween 80/saline solution was used as vehicle. The TRPV4 antagonist or vehicle was instilled into the SLN-innervated swallowing-related region for 15 min and then aspirated before applying GSK1016790A. Pilot experiments determined the lowest effective concentration of RN-9893 that reduced the 250 μM GSK1016790A-induced swallowing reflexes to ≤50% was 10 mM. Therefore, we used 10 mM RN-9893 to test the effect of a TRPV4 antagonist on GSK1016790A-induced swallowing reflexes.