Supplementary Figure 1. Inflammatory mediators on day 2 after acetic acid (AA) treatment in saline and 5-fluorouracil (5-FU)-treated rats. (A) No effects of indomethacin treatment (Indo) on the numbers of colony-forming units (CFUs) in saline- or 5-FU-treated rats. N=5 for each bar. (B) Cyclooxygenase-2 (COX-2) expression levels in the oral mucosa. N=5 for each bar. (C‒E) Interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α), respectively, levels in the oral mucosa. N=5 for each bar. *, ** and $, $$, P<0.05 and 0.01, respectively, compared with saline- treated and naïve rats, respectively; Student’s t test.
Supplementary Figure 2. Determination of the appropriate concentration of QX-314 using the intraoral dropping method on day 2 after acetic acid (AA) treatment. (A) Mouth rubbing during the 3 min following application of 1% and 3% QX-314 in water. Water was pre-applied as a control 20 min prior to the QX-314 application. No significant differences in mouth rubbing periods were found between 1% QX-314 and water (n=6), while 3% QX-314 evoked significantly more rubbing than water (*, P<0.05; Student’s t test, n=6). (B) Effect of the non-selective TRP channel blocker ruthenium red (RuR) on 3% QX-314-evoked mouth rubbing. The mouth rubbing measurements were performed 1 hr after topical treatment with RuR or vehicle (Veh, water). In the Veh treatment, 3% QX-314 application prolonged the mouth rubbing period compared with water application (*, P<0.05; Student’s t test, n=8). However, in the RuR treatment, there was no significant behavioral difference between the 3% QX-314 and water treatments (n=5),
suggesting that the higher dose of QX-314 elicits a pungent effect through the TRP channels.
Therefore, we used 1% QX-314 as a non-pungent anesthetic in the present study.
Supplementary Figure 3. Double-immunofluorescence for TRPA1 and TRPV1 in the trigeminal ganglion (TG) of naïve rats. (A) Representative immunofluorescent images for TRPA1 (green), TRPV1 (red) and merged (yellow). Arrows, double arrowheads and arrowheads indicate TRPV1-immunopositive (TRPV1+) alone, TRPA1-immunopositive (TRPA1+) alone and co- immunopositive, respectively. Scale bar, 100 μm. (B) Distributions of TRPA1 and TRPV1
expression by cell size. TRPV1- and TRPA1- represent immunonegative to TRPV1 and TRPA1,
respectively. (C) Summary of TRPA1- and TRPV1-immunoreactivities in small, medium and large neurons.