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. 2020 Nov 15;31(24):2687–2702. doi: 10.1091/mbc.E20-03-0200

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© 2020 Le et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 5: — SLC45A2 is required for melanosomes to progress from stage III to stage IV. (a–d) Melan-Ink4a (a), melan-uw (b), melan-p1 (c), and melan-uw cells stably expressing HA-SLC45A2 (melan-uw:HA-SLC45A2; d) were fixed and analyzed by conventional transmission electron microscopy. Insets of boxed regions on the left were magnified 4.5× on the right. I/II, stage I/II melanosomes; III, stage III melanosomes; IV, stage IV melanosomes. Scale bars, 500 nm (left) and 200 nm (right). (e) The number of stage I/II, stage III, and stage IV melanosomes per cell was quantified for each of the cell lines and is shown as a percentage of total counted melanosomes per cell. For each stage, data represent the mean ± SEM from the following number of independent experiments, cells, and melanosomes: melan-Ink4a (2, 54, 717), melan-uw (3, 49, 1292), melan-p1 (2, 27, 626), and melan-uw:HA-SLC45A2 (3, 76, 1927). Statistical analyses were performed by Welch’s ANOVA with multiple comparisons to melan-Ink4a for each stage and Dunnett T3 test for multiple comparisons. (f) Melanin content in cell lysates was determined by spectrometry relative to total protein content. Plat-E cells are included as a nonpigmented cell CTRL. The data are presented as a percentage normalized to melanin content in melan-uw samples ± SEM and represent at least three independent experiments performed in duplicate or triplicate. Statistical significance was determined by Welch’s ANOVA with Dunnett T3 test for multiple comparisons. Except where indicated, statistical comparisons were made relative to melan-uw. Note that all cells except melan-Ink4a and Plat-E were treated with 200 pM cholera toxin. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no significant difference.