COMMD3 deficiency results in α-granule protein following a degradative pathway, and the COMMD3 protein localizes to Stx12-positive endosomal subdomains. (A) Immunofluorescence microscopy analysis of WT and COMMD3 KO imMKCL cells treated with either vehicle (DMSO) or BafA1 and costained with P-selectin and PF4 antibodies. Bar = 5 µm. (B) Fluorescence intensity quantification of the cells shown in (A). Statistical significance was determined via unpaired, Mann-Whitney U test (WT DMSO, n = 27; WT BafA1, n = 35; COMMD3 KO DMSO, n = 38; COMMD3 KO BafA1, n = 57). A.U., arbitrary units. (C) Spinning disk confocal microscopy analysis of a COMMD3 KO imMKCL cell expressing GFP-COMMD3 and BFP-Stx12. Bar = 5 µm. (D) Quantification of BFP-Stx12 and GFP-COMMD3 colocalization of the cells shown in C (n = 25). (E) Left, superresolution scanning confocal Airyscan image of a COMMD3 KO imMKCL cell expressing GFP-COMMD3 and Cherry-Stx12 (bar = 5 µm). Right, multichannel view magnification of the region indicated by the dashed line rectangle (bar = 1 µm). The filled triangles indicate endosomal subdomains where GFP-COMMD3 and Cherry-Stx12 colocalize, while open triangles point toward Cherry-Stx12 endosomal tubules, which are COMMD3-negative but are continuous with GFP-COMMD3-positive subdomains. Supplemental Figure 16 shows additional examples of superresolution scanning confocal Airyscan images of COMMD3 KO imMKCL cells expressing GFP-COMMD3 and Cherry-Stx12.