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. Author manuscript; available in PMC: 2017 Jul 1.
Published in final edited form as: Stem Cells. 2016 May 3;34(7):1742–1752. doi: 10.1002/stem.2379

Figure 2.

Figure 2

Expression of polysialyltransferases is lineage specific and NCAM is the substrate of polysialylation. (A-C) qPCR expression of PST, STX, and NCAM (Top) and lineage markers (Bottom) in hPSC differentiation to DE (A), mesoderm (B), and NCC (C). (D) Immunostaining of PSA expression in DE day 4 cells transduced with shRNA targeting PST (Right) compared to control shRNA (Left). Scale bar, 100μm. (E) Flow cytometry of PSA and NCAM surface expression in hPSC, DE, and mesoderm. (F) Immunostaining of shRNA knockdowns of PST and NCAM in DE day 4. Scale bar, 100μm. (G) Immunostaining of PSA and α-mannosidase II expression in DE cells transduced with NCAM shRNA. Arrows point to overlap in expression. Scale bar, 100μm. (H) Immunoblot of NCAM (Top) and PSA (Middle) in all three germ layers. β-Actin shown as loading control (Bottom). (I) Immunoprecipitation of NCAM and PSA in hPSC and DE. IP lysate was then immunoblotted for PSA (Left) and NCAM (Right). Lysate from COS-1 cells overexpressing PST, STX, and NCAM was used as positive control. Abbreviations: hPSC, human pluripotent stem cell; DE, definitive endoderm; CTL, control.