Depletion of either PI4KIIα or PI4KIIβ by shRNA in WT melanocytes impairs localization of BLOC-1-dependent cargoes to melanosomes. (A–F) WT melan-Ink4a mouse melanocytes were transduced with lentiviruses to express shNC or the indicated shRNAs to PI4KIIα, PI4KIIβ, or the Pallidin subunit of BLOC-1 and selected for 7–9 d (A–E) or 14 d (F). Cells in D and E were additionally transiently transfected to express HA-tagged OCA2 (OCA2-HA) 2 d prior to analysis. (A and D) Cells were analyzed by IFM for TYRP1 (A; green, left and right panels) or HA (D; left and right panels), and by bright field microscopy (BF) for pigment granules (middle; pseudocolored magenta at right). White arrows, pigment granules surrounded by TYRP1 or overlapping with OCA2-HA; yellow arrowheads, TYRP1 or OCA2-HA not associated with pigment granules. Boxed regions are magnified fivefold in insets. Scale bars: main panels, 10 μm; insets, 2 μm. (B, C, and E) Quantification of TYRP1 (B) or HA-OCA2 (E) overlap with melanosomes or melanosome coating by TYRP1 (C). Data from three independent experiments each were analyzed by Kruskal–Wallis (B and C) or Welch’s ANOVA (E); ****, P < 0.0001. (F) shRNA-treated melan-Ink4a cells or HEK293T cells as a negative control were analyzed by quantitative melanin content assay. Data are presented as mean ± SD of five independent experiments as a percentage of the signal from shNC-treated cells and were analyzed by Welch’s ANOVA relative to shNC-treated cells; ****, P < 0.0001; *, P < 0.05.