Figure 3. Assessment of MK maturation and platelet formation.

(A) Confocal analyses of vWF and CD63 expression in MK (left panel) and colocalization analyses (right panel). Scale bars: 50 μm. (B) Schematic overview of the normal development steps of DMS. (C) Confocal analysis of CD41 marker distribution in iMKs. Scale bars: 50 μm. (D) Ultrastructural characterization of the iMK according to the compared genotypes. A representative MK is shown for each condition (upper panels), a part of which (dotted square) is enlarged (lower panels). More than 80% of T and TS MKs showed maturity characteristics associated with condensed nuclei, a well-developed DMS (blue arrows), and the presence of normal α-granules (red arrows). More than 90% of TG, TGM, and TGS MKs showed blockages in maturation characterized by the presence of uncondensed nuclei majorly composed of euchromatin and the absence of DMS formation, with endosomes presenting an abnormal accumulation of granules (red arrows). Scale bars: 5 μm. (E) Representative microphotographs of CD41⁺CD42⁺ iMKs under PPT formation assay. PPT-forming MKs are highlighted with blue arrows in the T and TS conditions. Scale bars: 50 μm. (F) Histogram of the number of PPT-forming MKs in the compared conditions. Data are represented as mean ± SEM; n = 3–4. The number of clones tested per genotype was as follows: T/parental = 1; TS = 3; TG = 2; TGM = 4; TGS = 3. Statistical significance was determined using 1-tailed Mann-Whitney’s U test. *P < 0.05; **P < 0.01; ***P < 0.001.