Fig. 4.

Bitter (T2R) agonists activate Ca²⁺ _i responses in cultured HNSCC. HNSCC cell line SCC47 loaded with Ca²⁺ binding dye, Fluo‐4, was stimulated with T2R agonists, and Ca²⁺ was measured over time. (A) Peak Fluo‐4 F/F_o was quantified after stimulation with Hank's Balanced Salt Solution (HBSS), denatonium benzoate, sodium benzoate, quinine, diphenidol, flufenamic acid, parthenolide, thujone, 3‐oxo‐C12HSL, phenylthiocarbamide (PTC), and purinergic receptor agonist adenosine triphosphate (ATP) (mean ± SEM; 3 experiments using separate cultures). Significance by 1‐way ANOVA with Bonferroni posttest comparing HBSS to each agonist. (B) Table of T2R targets for agonists used. Effective concentration (EC, in µm) or half‐maximal effective concentration (EC₅₀, when indicated) shown in parentheses, taken from [40, 50]. Compounds with ‘ND’ denote EC not determined. (C, D) Representative traces of Fluo‐4 F/F_o over time after stimulation with denatonium benzoate (C) and sodium benzoate (separate traces superimposed) and quinine (D). Traces are from n = 1 culture each but are representative of results obtained from 3 independent cultures (mean ± SEM shown in A). (E) Fluo‐4 fluorescence in response to denatonium benzoate‐induced Ca²⁺ release appears to localize more strongly to the nucleus than the cytoplasm as represented by the traces and images; peak Fluo‐4 F/F_o for nuclear Ca²⁺ release is significantly greater than cytoplasmic Ca²⁺ release (mean ± SEM; 3 separate cultures). Significance by unpaired t‐test. Scale bar is 10 µm. (F, G) Nuclear‐localized R‐GECO‐nls was used to measure nuclear Ca²⁺ after stimulation with (F) denatonium benzoate and (G) quinine. Both agonists stimulate nuclear Ca²⁺ as demonstrated by the traces over time and peak GECO F/F_o compared to HBSS (mean ± SEM; 3 separate cultures). Significance by unpaired t‐test. *P < 0.05; **P < 0.01.