PPF by CLEC-2 WT megakaryocytes is facilitated by coculture with BM FRC-like cells. (A) Representative ICC images of proplatelet-forming megakaryocytes in coculture with BM FRC-like cells. CLEC-2 WT megakaryocytes (left panel) and CLEC-2 cKO megakaryocytes (right panel) were cocultured with BM FRC-like cells for 5 days. Megakaryocytes were stained with CD41 (red). Arrowheads indicate the proplatelet-forming megakaryocytes. Scale bar, 50 μm. (B-C) Quantification of PPF megakaryocytes in a fetal liver PPF model, with or without recombinant PDPN (PDPN-rFC2) treatment. (B) Cell-autonomous PPF in CLEC-2 WT (n = 3) and cKO megakaryocytes (n = 5). (C) Stimulated PPF with PDPN-rFC2 (20 μg/mL) in CLEC-2 WT and cKO megakaryocytes. CLEC-2 WT with rFC2, n = 3; CLEC-2 WT with PDPN, n = 3; CLEC-2 cKO with rFC2, n = 6; CLEC-2 cKO with PDPN, n = 7. (D) Scheme of the conditioned medium used in the fetal liver PPF assays. (E) Quantification of PPF with rFC2-conditioned medium or CLEC-2–conditioned medium, n = 6 per group. The error bars represent SEMs. ***P < .001. N.S., a nonsignificant difference.