DNM2 and DNM3 are expressed in human MKs and modify plasma membrane anisotropy. (A) OMX super-resolution microscopy image of DNM2 (red) and DNM3 (green) staining in human MKs; original magnification, 63× objective. Twenty-five or more primary MKs were analyzed by confocal immunofluorescence. Here shown are OMX super-resolution microscopy images for illustration purposes. DNM2 specks are distinct from the DNM3 dotted staining, and both are distributed throughout the cytoplasm. Blue stains for the nucleus. Scale bar represents 2 µm. (B) Western blot (WB) of DNM2 and DNM3 and efficiency of their knockdown in CHRF cells. DNM2 presented as a single band in the megakaryocytic CHRF cells, whereas DNM3 migrated as 2 bands: a band at ∼90 kDa corresponding to the classical transcript, along with a shorter band, corresponding to an alternate DNM3 transcript (∼75 kDa). The fold change of DNM expression in shRNA-induced knockdown, compared with control (CTRL) cells (nontarget CTRL shRNA), is indicated as a percentage of CTRL. (C) WB staining of DNM3 in CHRF cells and MKs. The presence of the alternate (Alt) DNM3 band (arrow) is confirmed in primary human MKs (hMKs) differentiated from cord blood CD34 cells. (D) Plasma membrane fluorescence anisotropy measurement in CHRF cells with DNM2 and DNM3 knockdown. The measurement of plasma membrane fluorescence anisotropy values (r) in response to activation by PMA (1 µM) indicated a trend for decreased membrane fluidity, deformation, and remodeling for cells with single DNM knockdown and showed significant difference from CTRL for cells with double shDNM2 and shDNM3 knockdown (at 30 minutes). The stronger effect with double knockdown suggested that the actions of DNM2 and DNM3 overlap to a certain degree. Error bars indicate standard errors of the mean of ≥3 independent experiments. **P ≤ .01, ***P ≤ .001. MW, molecular weight.