Fig. 1
Increased intracellular Ca2+ concentrations were observed after application of FK506 in HEK293T cells expressing human TRPA1 and TRPM8. a–g Representative changes in intracellular Ca2+ concentrations ([Ca2+]i) stimulated by 30 µM FK506 in mock-transfected HEK293T cells (a), or HEK293T cells expressing human TRPV1 (hTRPV1, b), TRPV2 (hTRPV2, c), TRPV3 (hTRPV3, d), TRPV4 (hTRPV4, e), TRPA1(hTRPA1, f) or TRPM8 (hTRPM8, g). To confirm the expression of each TRP channel, 1 μM capsaicin (Cap), 500 μM 2-aminoethyl diphenylborinate (2APB), a mixture of 250 μM 2APB and 500 μM carvacrol (Mix), 300 nM GSK1016790A (GSK), 100 μM allyl isothiocyanate (AITC), or 500 μM menthol (Men) was used. Cell viability was checked by application of 5 μM ionomycin (Iono). Y-axis: Fura-2 ratio (340 nm/380 nm). Each symbol represents mean ± SEM from 33 to 105 cells. h Summary of [Ca2+]i increases following application of 30 μM (white column) or 55 μM (black column) of FK506 to HEK293T cells expressing hTRPV1, hTRPV2, hTRPV3, hTRPV4, hTRPA1 or hTRPM8. Y axis: ΔRatio calculated by the normalization of peak Fura-2 ratio (340 nm/380 nm) of FK506 to that of ionomycin. “Mock” indicates the [Ca2+]i increases in HEK293T cells transfected with empty vector. Each column represents the mean + SEM from 22 to 184 cells. Statistical significance was assessed using ANOVA followed by a two-tailed multiple t test with Bonferroni correction. **, P < 0.01 vs. mock