Figure 5.
COMMD3 defines a major form of the CCC complex in MK cells and is required for normal α-granule biogenesis. (A) RNAseq analysis for the 10 COMMD genes indicates COMMD3, COMMD5, and COMMD7 are more highly expressed in differentiated than in undifferentiated imMKCL cells. (B) IB analysis of total extracts of undifferentiated and differentiated imMKCL cells. (C) Quantification of the results obtained in (B). Differentiated samples were compared with undifferentiated samples, and statistical significance was determined via unpaired, 2-tailed Student t test (n = 2). (D) Total extracts of WT or COMMD3 KO imMKCL cells were prepared and subjected to IB analysis with the indicated antibodies. Three independent experiments per cell type are shown. (E) Quantification of the results obtained in (D) expressed as the ratio of the normalized band intensities of P-selectin or PF4 in COMMD3 KO cells relative to WT imMKCL cells. COMMD3 KO samples were compared with WT samples, and statistical significance was determined via unpaired, 2-tailed Student t test (n = 3). (F) Immunofluorescence microscopy analysis of WT or COMMD3 KO cells costained with P-selectin and EEA1 antibodies. Bar = 5 µm. (G) Fluorescence intensity quantification of the cells shown in (F). COMMD3 KO samples were compared with WT samples, and statistical significance was determined via unpaired, Mann-Whitney U test (WT, n = 26; COMMD3 KO, n = 25). A.U., arbitrary units. (H) Thin-section transmission electron microscopy analysis of WT and COMMD3 KO imMKCL cells subjected to high-pressure freezing. Original magnification, ×11 000 for the picture showing the entire cell and ×49 000 for the enlarged insets (bars represent 1 μm and 100 nm, respectively). (I) Quantification of α-granules, δ-granules, and MVBs visualized in the experiment shown in (H). COMMD3 KO samples were compared with WT samples, and statistical significance was determined via unpaired, Mann-Whitney U test (WT n = 12; COMMD3 KO n = 13 cells). The WT data set is the same as the one shown in Figure 2F. ν, nucleus; α, α-granules; δ, δ-granules; MVB, multivesicular body.

COMMD3 defines a major form of the CCC complex in MK cells and is required for normal α-granule biogenesis. (A) RNAseq analysis for the 10 COMMD genes indicates COMMD3, COMMD5, and COMMD7 are more highly expressed in differentiated than in undifferentiated imMKCL cells. (B) IB analysis of total extracts of undifferentiated and differentiated imMKCL cells. (C) Quantification of the results obtained in (B). Differentiated samples were compared with undifferentiated samples, and statistical significance was determined via unpaired, 2-tailed Student t test (n = 2). (D) Total extracts of WT or COMMD3 KO imMKCL cells were prepared and subjected to IB analysis with the indicated antibodies. Three independent experiments per cell type are shown. (E) Quantification of the results obtained in (D) expressed as the ratio of the normalized band intensities of P-selectin or PF4 in COMMD3 KO cells relative to WT imMKCL cells. COMMD3 KO samples were compared with WT samples, and statistical significance was determined via unpaired, 2-tailed Student t test (n = 3). (F) Immunofluorescence microscopy analysis of WT or COMMD3 KO cells costained with P-selectin and EEA1 antibodies. Bar = 5 µm. (G) Fluorescence intensity quantification of the cells shown in (F). COMMD3 KO samples were compared with WT samples, and statistical significance was determined via unpaired, Mann-Whitney U test (WT, n = 26; COMMD3 KO, n = 25). A.U., arbitrary units. (H) Thin-section transmission electron microscopy analysis of WT and COMMD3 KO imMKCL cells subjected to high-pressure freezing. Original magnification, ×11 000 for the picture showing the entire cell and ×49 000 for the enlarged insets (bars represent 1 μm and 100 nm, respectively). (I) Quantification of α-granules, δ-granules, and MVBs visualized in the experiment shown in (H). COMMD3 KO samples were compared with WT samples, and statistical significance was determined via unpaired, Mann-Whitney U test (WT n = 12; COMMD3 KO n = 13 cells). The WT data set is the same as the one shown in Figure 2F. ν, nucleus; α, α-granules; δ, δ-granules; MVB, multivesicular body.

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