Fig. 3: Caveolae contributes to changes in melanocyte morphology, contacts with keratinocytes and mechanoprotection.

a IFM images of melanocytes treated with Ctrl (control) or Cav1 (caveolin-1) siRNA and incubated for 14 h with poor medium (+DMSO), supplemented medium (+DMSO), poor medium + FSK (forskolin, 30 µM) or Ker-CM (keratinocytes-conditioned medium) for 14 h, immunolabelled for Cav1 (caveolin-1, green) and stained for F-actin (phalloidin, red). Cav1 polarization, arrowheads; cell protrusions, asterisks. Bars, 20 µm. b Frequency of melanocytes [as in a] showing at most two (≤2) or more than two (>2) membrane protrusions (n = 150 cells, three independent experiments). c Quantification of the length-to-width ratio of melanocytes cultured as in a. d, e Melanocytes treated for 72 h with siCtrl or siCav1 were co-cultured with keratinocytes for 14 h prior to cell imaging. d Representative projection of time-lapse images with interpolated region of interest for the cell’s boundaries every 20 min. Bars, 10 µm. See also Supplementary Videos 2 and 3. e Frequency of keratinocytes contacting melanocytes for a total of 4 h. f Immunoblot analysis of p-MLC (phosphorylated myosin light chain) and MLC (myosin light chain) levels in siCtrl- and siCav1-treated melanocytes maintained for 4 h in poor medium, supplemented medium or poor medium + FSK. g Quantification of MLC activation in f, corresponding to the ratio of p-MLC on MLC total expression level after GAPDH (glyceraldehyde 3-phosphate dehydrogenase) normalization. h, i Melanocytes treated with siCtrl or siCav1 for 72 h were incubated with calcein-AM (green) for 15 min, washed and subjected to hypo-osmotic shock (30 mOsm) in the presence of PI (propidium iodide, red) for 10 min. PI-positive cells (red nuclei) indicate melanocytes with ruptured plasma membrane. See also Supplementary Videos 6 and 7. h First (0 min) and last (10 min) still images from the time-lapse acquisition. Bars, 50 µm. i Frequency of bursting melanocytes. Values are the mean ± s.e.m. b, c, e Three independent experiments. i siCtrl, n = 714 cells, siCav1, n = 958 cells; three independent experiments. g Six independent experiments. b, c One-way ANOVA with Tukey’s multiple-comparison test; e two-tailed paired t-test; g one-way ANOVA with Dunnett’s multiple-comparison test; i two-tailed unpaired t-test with Welch’s correction. See also Supplementary Table 3.