Assessment of lineage markers in control, LGR4, LGR5, and LGR4/5 knockdown hESC lines. h9–shRNA–scrambled, h9–shRNA–LGR4, h9–shRNA–LGR5, and h9–shRNA–LGR4/5 knockdown lines all were treated for 3 days with ACTA to induce endoderm formation. qRT-PCR showing mRNA expression above hESCs for (A) neural markers PAX6 and NESTIN, and (B) mesenchymal lineage markers BRACHYURY, VIMENTIN, and FOXF1. One-way analysis of variance was used for statistical analysis. *P ≤ .05, **P ≤ .01, ***P ≤ .001, and ****P ≤ .0001.