ST3GAL1 and ST3GAL2 modify multiple glycoproteins in HPCs and outcompete the formation of core 2 O-glycans on CD34 and CD43. (A) Flowcytometric analysis showed production of CD34⁺ HPCs from different iPSC lines. (B) Quantitated yield of CD34⁺ HPCs from different iPSC lines. Values represent the means plus or minus SEM from 3 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 HPCs. Numbers indicate median fluorescence intensities. (D) PNA blot revealed multiple glycoprotein substrates for ST3GAL1 and ST3GAL2 in lysates from HPCs. (E) Schematic of O-glycan biosynthesis and plant lectin binding epitopes. (F) Pull-down experiment using PNA-agarose beads from lysates of iPSC-derived HPCs, followed by SDS-PAGE and immunoblot with either PNA, STL lectins or sheep anti-CD34 or goat anti-CD43 polyclonal antibodies. (G) Western blot analysis of CD34 and CD43 expression in whole-cell lysates from iPSC-derived HPCs. SDS-PAGE, SDS-polyacrylamide gel electrophoresis.