Fig. 5. Calcium chelation or PLC inhibition prevent MC1R-mediated enhancement of HAoEC migration. (A) After insert removal, HAoEC (no. c2) monolayers were treated with α-MSH 10^-8 M, with or without pre-treatment with the intracellular Ca²⁺ chelator BAPTA-AM 10^-5 M, and allowed to migrate for 3, 9, and 12h: gap closure was quantified using DiR cell staining and near-infrared fluorescence scanning. Results are shown as mean ± SEM (n = 5-6 per group). Statistical significance was assessed by two-way ANOVA [F_(9,68) = 2.337, p=0.0233, interaction time × treatment; F_(3,68) = 5.493, p=0.0019, treatment effect] with Bonferroni post-hoc test [*p<0.05, **p<0.01, α-MSH vs. medium alone (C)]. (B) Directional migration assay was carried out also pre-treating cells with the PLC inhibitor U-73122. Results are shown as mean ± SEM (n = 5-6). Statistical significance was assessed by two-way ANOVA [F_(9,68) = 2.607, p=0.0120, for interaction; F_(3,68) = 7.102, p=0.0003, treatment effect] with Bonferroni post-hoc test (*p<0.05).