Fig 1: Rab27a and Mlph disperse melanosomes in melan-R3GKO cells. (A) Coomassie-stained gel and western blots of cell lysates showing protein loading and Rab27a and Mlph expression. (B–D) melan-R3GKO cells were transfected with Rab27a-specific and non-targeted (NT) control siRNA, and the effect on protein expression and melanosome distribution investigated. (B) Western blot showing Rab27a and GAPDH (loading control) expression in lysates of melan-R3GKO non-transfected (mock) or siRNA-transfected cells. (C) Phase-contrast images showing the melanosome distribution in transfected melan-R3GKO cells. Boxes in left panels indicate the area shown in higher magnification on the right. (D) Scatter plot showing the percentage of clustered-type cells in melan-R3GKO cultures 72 h after transfection. Data are from four independent experiments each performed in triplicate. Plotted points represent the mean data from each experiment, data presented as mean±s.e.m. (E–G) Melanocytes were fixed, stained for immunofluorescence and the distribution of Mlph and melanosomes recorded using a confocal microscope. (E) Fluorescence (left), phase-contrast (centre) and merged images showing the distribution of Mlph, melanosomes and their overlap. White boxes indicate the regions shown in high magnification images below. White arrowheads indicate co-localisation of melanosomes and Mlph. (F,G) Scatter plots showing the mean anti-Mlph fluorescence intensity/cell for different cell types (F), and clustered- and dispersed-type melan-R3GKO cells (G). Results presented are representative of three independent experiments. ****P<0.0001, **P<0.01, *P<0.05. Scale bars: 20 µm.