Reinforced expression of active Rab32 suppresses the defects in melanosome biogenesis and melanin production in B16F10 cells with shD1/D2. (A) Overexpression of mCh-Rab32(WT) and mCh-Rab32(Q85L), but not mCh-Rab32(T39N), suppresses melanosome enlargement in cells treated with shD1/D2. Melanosomes were labeled with GFP-Tyrosinase. Representative images (left) and quantification of the diameters of melanosomes (right) are shown. The boxed region is magnified (3×) at the bottom right in each image. For each form of Rab32, the corresponding merged images (GFP-TYR+mCh-Rab32) are magnified (6×) and shown in the right column. Scale bars, 5 μm. Diameters of 80 melanosomes were measured in each group (mean ± SEM, n = 80 melanosomes, unpaired two-tailed t test, ***P < 0.001, ns: not significant). (B) Overexpression of mCh-Rab32 (WT) and mCh-Rab32 (Q85L), but not mCh-Rab32 (T39N), restores melanin production in B16F10 cells treated with shD1/D2. Representative images (left) and melanin production was quantified by calculating A405/A562 (right) (mean ± SEM, n = 4 independent experiments, unpaired two-tailed t test, ***P < 0.001, ns: not significant). EV, empty vector. (C and D) Overexpression of mCherry-HPS1 or mCherry-HPS4 does not rescue melanosome enlargement (C) or melanin production (D) in B16F0 cells with shD1/D2. Representative fluorescence images are shown in C. Scale bars, 5 μm. (D) Representative images of melanin (left) and melanin production was quantified by calculating A405/A562 (right) (mean ± SEM, n = 4 independent experiments, unpaired two-tailed t test, ***P < 0.001; ns: not significant). (E) Graphic summary of the roles of the LMD-2–(GLO-3–CCZ-1)–GLO-1 axis in promoting LRO biogenesis in C. elegans and the LYSMD1/2–(HPS1–HPS4)–Rab32 axis in promoting melanosome biogenesis in mammals.