Loss of ST3GAL1 and ST3GAL2 disrupts O-glycan sialylation of major surface glycoproteins on MKs and impairs proplatelet formation. (A) Flow cytometric analysis showed production of CD41⁺/CD42b⁺ MKs from different iPSC lines. (B) Quantitated yield of CD41⁺/CD42b⁺ MKs from different iPSC lines. Values represent the means plus or minus SEM from 4 independent experiments. No significance was found by 1-way ANOVA with Dunnett’s test compared with WT cells. (C) Flowcytometric analysis of PNA binding to iPSC-derived MKs. Numbers indicate median fluorescence intensities. (D) PNA blot revealed a subset of glycoprotein substrates for ST3GAL1 and ST3GAL2 in lysates from MKs. (E) Immunoprecipitation with anti-GPIbα monoclonal antibody AP1 from lysates of iPSC-derived MKs, followed by SDS-PAGE and immunoblot with either PNA or rabbit anti-GPIbα polyclonal antibody. Immunoprecipitated GPIbα from different cell lines was treated with neuraminidase for complete exposure of PNA-reactive epitopes as positive controls. (F) Coimmunoprecipitation of GPIIb-IIIa complex with anti-GPIIIa monoclonal antibody AP3 from lysates of iPSC-derived MKs, followed by SDS-PAGE and immunoblot with either PNA or rabbit anti-GPIIb polyclonal antibody. (G) Immunofluorescence staining of iPSC-derived MKs cultured on fibrinogen-coated glass slides. The cells were labeled with phalloidin (red), anti-GPIbα antibody (green), and 4′,6-diamidino-2-phenylindole (blue). Scale bar, 20 μm. White arrows indicate proplatelet-forming MKs. (H) Quantitated percentage of proplatelet-forming MKs from each cell line. Values represent the means plus or minus SEM from 3 independent experiments. *P < .05 by 1-way ANOVA with Dunnett’s test compared with WT cells. n.s., not significant; SDS-PAGE, SDS-polyacrylamide gel electrophoresis.