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. 2013 Oct 10;2(11):819–829. doi: 10.5966/sctm.2013-0069

Figure 4.

Figure 4.

Influence of PIG-A mutations in early development. (A): Relative expression of germ layer markers by quantitative reverse transcription-polymerase chain reaction. By day 3 of differentiation, embryoid bodies (EBs) of both hFib2-iPS5 and FPHR-PIG-A cells showed upregulation of early mesodermal markers BRACHYURY and MIXL1 compared with FPHR cells. Endodermal markers FOXA2 and SOX17 were similarly upregulated at days 3 and 7 in both hFib2-iPS5 and FPHR-PIG-A compared with FPHR cells. Conversely, ectodermal markers PAX6 and SOX1 remained repressed throughout differentiation in both hFib2-iPS5 and FPHR-PIG-A cells but were upregulated in FPHR cells. (B): Western blot analysis for BMP signaling activation of hFib2-iPS5 and FPHR cells. All cells were treated with/without BMP-4. After 6 hours of BMP-4 induction, phosphorylated Smad 1/5/8 (60 kDa) was detected using specific phosphorylated Smad 1/5/8 antibody, and phosphorylated Smad 1/5/8 was significantly induced in hFib2-iPS5 cells but not FPHR cells. β-Actin was used as endogenous control. (C): Quantitative analysis of the intensity of fold change in hFib2-iPS5 and FPHR cells. The expression of phosphorylated Smad 1/5/8 was not detected in FPHR cells without BMP-4 and slightly increased in FPHR but significantly lower than hFib2-iPS5 cells after BMP-4 induction. *, p < .05. Abbreviations: BMP, bone morphogenetic protein; iPS, induced pluripotent stem cell.