GALE p.Lys78ValfsX32, p.Thr150Met, p.Val128Met, and p.Leu223Pro variants impaired actin and filamin A distribution among the MK cytoplasm. MKs were differentiated from peripheral blood progenitor cells (probands A.II.4 and B.II.2) through a 14-days culture, in parallel with healthy controls. (A) Representative image of MKs stained with phalloidin (green) and anti-filamin A (red) antibodies in control, A.II.4, and B.II.2 samples. We observed a heterogeneously disorganized and nonuniform actin cytoskeleton, based on the presence of actin filaments distributed in patches (white asterisk), a heterogeneous delocalization of filamin A with actin, and the presence of actin in clusters with reduced levels of filamin A (white arrow). (B) Representative image of MKs stained with an anti-β1–tubulin to investigate microtubule assembly in MKs forming proplatelets. Hoechst (blue) was used for counterstaining nuclei. Scale bars, 20 μm. (C) Immunoblotting of Mk lysates. Control, A.II.4, and B.II.2 samples were probed with anti-β actin and anti-β3 integrin (internal control).