Figure 1.

STIM1 expression and activity increases with melanogenesis.A, representative B16 cell pellet pictures of LD day 0, LD day 4, and LD day 7. B, qRT–PCR analysis showing increase in STIM1 mRNA expression with pigmentation in B16 LD model (N = 3). C, representative Western blot showing an increase in STIM1 protein expression on LD day 7 in comparison with day 0 (N = 3). D, densitometric quantitation showing increase in STIM1 protein levels on LD day 7 in comparison with day 0 (N = 3). E, representative pellet pictures upon αMSH treatment in B16 cells as compared to control (N = 3). F, qRT–PCR analysis showing increase in STIM1 mRNA expression in B16 cells upon αMSH treatment (N = 3). G, representative Western blot showing STIM1 protein levels in B16 cells upon 1 μM αMSH treatment for 48 h as compared to vehicle control (N = 4). H, densitometric quantitation showing increase in STIM1 protein levels upon αMSH treatment (N = 4). I, representative Western blot showing Orai1 protein levels upon αMSH treatment in B16 cells as compared to vehicle control (N = 3). J, densitometric quantitation showing Orai1 protein levels upon αMSH treatment (N = 3). K, representative Western blot showing Orai2 protein levels upon αMSH treatment in B16 cells as compared to control (N = 3). L, densitometric quantitation showing Orai2 protein levels upon αMSH treatment (N = 3). M, representative Western blot showing Orai3 protein levels upon αMSH treatment in B16 cells as compared to control (N = 3). N, densitometric quantitation showing Orai3 protein levels upon αMSH treatment (N = 3). O, representative Ca²⁺ imaging trace of vehicle control where “n = 43” denotes the number of cells in that particular trace. Cells were stimulated with 2 μM thapsigargin (Tg) in Ca²⁺-free buffer followed by restoration of 2 mM extracellular Ca²⁺. P, representative Ca²⁺ imaging trace of αMSH treatment where “n = 41” denotes the number of cells in that particular trace. Cells were stimulated with 2 μM thapsigargin (Tg) in Ca²⁺-free buffer followed by restoration of 2 mM extracellular Ca²⁺. Q, the extent of SOCE was calculated from 119 Control and 117 αMSH-treated B16 cells, which were imaged from three independent experiments (“n = x, y” where “x” denotes total number of cells imaged and “y” denotes number of traces recorded). Data presented are mean ± S.E.M. For statistical analysis, one sample t test was performed for panels B, D, F, H, J, L, and N, and unpaired student’s t test was performed for panel Q using GraphPad Prism software. Here, NS means nonsignificant; ∗p <0.05 and ∗∗p < 0.01. αMSH, α-Melanocyte Stimulating Hormone; LD, low-density; SOCE, store-operated Ca2+ entry; STIM1, Stromal Interaction Molecule1.