Keratinocyte paracrine secreted factors ET-1 and ACh induce dendritic Ca²⁺ transients. (A) Ca²⁺ oscillations in melanocytes co-cultured with keratinocytes in response to 10 nM ET-1 (black line: addition of 10nM ET-1 to imaging bath); ΔF/F0 plots corresponding to numbered regions on the melanocyte. See also Video 3. (B) ET-1 increases the number of Ca²⁺ transients per co-cultured melanocyte via the ET_B receptor (n = 203, 141, 141, and 62 cells from 3, 2, 2, and 1 different patient-derived and matched co-cultures, respectively). Significant by Kruskal-Wallis ANOVA (H = 67.63, 3 degrees of freedom [d.f.], P = 1.37 × 10⁻¹⁴). Box: 25%, 75%; whiskers: 10–90%. (C) Co-cultured melanocyte dendritic Ca²⁺ transients induced by 10 nM ET-1. Top: Co-cultured melanocytes, expressing mCherry (shown) and GCaMP6f, before the addition of ET-1. Bottom: Kymograph (GCaMP6f) from corresponding color-coded line in top panel; black line denotes imaging media (left, “bath”) or change to imaging media containing 10 nM ET-1 (right, +10 nM ET-1). (D) Ca²⁺ spread from a single spontaneous dendritic transient before addition of ET-1, from > in kymograph in C. Thick line, full-width half maximum Gaussian fit. (E) Ca²⁺ spread from four dendritic transients after the addition of 10nM ET-1. Colors correspond to arrowheads with the same colors in the +10 nM ET-1 kymograph in C. Thick line, full-width half maximum Gaussian fit. (F) 100 µM ACh elicits local transient Ca²⁺ response in melanocyte dendrites. Black line: addition of ACh (black ΔF/F0 plot) or imaging media (bath, green ΔF/F0 plot). See also Video 5. (D) ACh, via mAChRs, increases the number Ca²⁺ transients per co-cultured melanocyte (n = 243, 128, 128, 115, and 131 cells from 4, 2, 2, 2, and 2 different patient-derived and matched co-cultures, respectively). Significant by Kruskal-Wallis ANOVA (H = 41.32, 4 d.f., P = 2.371 × 10⁻⁸). Box: 25%, 75%; whiskers: 10–90%. Scale bars, 100 µm.