DNMs regulate actin cytoskeleton polymerization, RhoA activation, and surface CXCR4 expression in human MKs. (A) Distribution of F-actin in MKs of vehicle-treated control (CTRL) and DNSR-treated MKs plated on FN and stained with phalloidin (red) and DAPI (blue), without and with DNSR treatment. Twenty-five or more primary MKs were analyzed by confocal immunofluorescence. Here shown are OMX super-resolution microscopy images for illustration purposes. DNSR-treated MKs showed more unevenly distributed, disorganized, and clumped F-actin (arrow) compared with CTRL cells; original magnification, 63× objective. Scale bar represents 2 µm. (B-C) F-actin polymerization in MKs without and with DNSR treatment. Flow cytometry representative histograms and quantification in 3 experiments using fluorophore-conjugated phalloidin staining in permeabilized and fixed MKs. Actin polymerization was decreased by ∼25% in DNSR-treated primary MKs (paired Student t test P ≤ .01). (D-E) F-actin polymerization in CHRF cells without and with DNM knockdown. Flow cytometry representative histograms and quantification in 4 experiments using fluorophore-conjugated phalloidin staining in permeabilized and fixed CHRF cells. CHRF with shDNM2 or double knockdown for DNM2 and DNM3 showed an ∼25% decrease in actin polymerization when compared with CTRL cells (P < .05). A similar trend was observed for CHRF cells with single shDNM3 knockdown. (F) RhoA activation quantification by G-LISA in MKs on FN. RhoA activation was reduced by half in DNSR-treated MKs (paired Student t test P < .05). (G-H) Surface CXCR4 expression in MKs. Flow cytometry representative histograms and quantification in 3 experiments. By flow cytometry, surface CXCR4 was slightly increased in DNSR-treated MKs (paired Student t test P < .05). Error bars indicate standard errors of the mean of ≥3 independent experiments. *P ≤ .05, **P ≤ .01. MFI, mean fluorescence intensity; OD, optical density.