Inhibition of endogenous endothelin and ACh signaling reduce melanocyte Ca²⁺ transients. (A) Antagonism of ET_B (BQ788) but not ET_A (BQ123) significantly reduces the number of melanocytes with Ca²⁺ transients. Control: vehicle. n = 6, 3, and 3 co-cultures, respectively derived from six different donors. See also Video 4. One-way ANOVA F(2,9) = 39.38, P = 0.000035; post hoc Tukey means comparison to other culture conditions: **, P = 0.05; data represented as mean ± SD. (B) Knockdown of melanocyte ET_B (EDNRB 13.1 and 13.2) reduced the number of melanocytes with Ca²⁺ transients in co-cultures. One-way ANOVA F(4,10) = 8.21, P = 0.0033; post hoc Tukey means comparison to other culture conditions, *, P < 0.05; data represented as mean ± SD. n = 3 co-cultures. (C) Relative expression level of EDN1, determined by qRT-PCR, in keratinocytes transduced with three different shRNA against EDN1 normalized to GFP control. n = 3 technical replicates. (D) EDN1 knockdown in keratinocytes reduces the number of co-cultured melanocytes, expressing RCaMP1h, with Ca²⁺ transients. n = 3 co-cultures for each condition. One-way ANOVA F(3,8) = 10.5, P = 0.0037; post hoc Tukey means comparison to other culture conditions, *, P < 0.05. (E) mAChR antagonist atropine reduces the number of co-cultured melanocytes with Ca²⁺ transients. Control: vehicle. n = 5, 3, and 3 co-cultures, respectively, derived from five different donors. One-way ANOVA F(2,8) = 28.75, P = 0.00022; post hoc Tukey means comparison, **, P < 0.005; *, P = 0.10. (F) Relative expression level of CHAT, determined by qRT-PCR, in keratinocytes transduced with two different shRNA against CHAT, normalized to GFP control. n = 3 technical replicates. (G) CHAT knockdown in keratinocytes reduced the number of co-cultured melanocytes, expressing RCaMP1h, with Ca²⁺ transients. n = 3 co-cultures for each condition. One-way ANOVA F(2,6) = 20.7, P = 0.0020; post hoc Tukey means comparison to other culture conditions, **, P < 0.005. All data represented as mean ± SD.