Skip to main content
. 2016 Jun 23;2(5):648–662.e8. doi: 10.1016/j.jcmgh.2016.06.002

Figure 6.

Figure 6

Exogenous RSPO1 rescues loss of LGR4 or LGR5 knockdown. (A) qRT-PCR mRNA abundance in all samples for FOXA2, SOX17, and AXIN2. h9–shRNA–scrambled, h9–shRNA–LGR4 knockdown, h9–shRNA–LGR5 knockdown, and h9–shRNA–LGR4/5 knockdown lines all were treated for 3 days with ACTA, or with ACTA + RSPO1 (500 ng/mL) to induce endoderm formation. LGR4, 5, and 4/5 knockdown lines treated with RSPO1 showed a statistically significant rescue of expression levels for all genes in all knockdown lines, except for AXIN2 in the LGR4 knockdown line. One-way analysis of variance was used for statistical analysis across groups. (B) h9–shRNA–scrambled, h9–shRNA–LGR4 knockdown, h9–shRNA–LGR5 knockdown, and h9–shRNA–LGR4/5 knockdown lines all were treated for 3 days with ACTA to induce endoderm formation or with ACTA + RSPO1, and immunostained for FOXA2 (red) and SOX17 (green). Nuclei were stained with DAPI (blue). Scale bars represent 200 μm. (C) Cell staining shown in panel B was quantitated. Each color group, corresponding to the percentage of FOXA2+ cells (red), the percentage of SOX17+ cells (green), and the percentage of double-positive cells (blue) was compared using 1-way analysis of variance. Statistical comparisons were performed between a particular knockdown group and the corresponding RSPO1-treated group (ie, the percentage of FOXA2-positive in shRNA–LGR5 vs shRNA–LGR5 + RSPO1, and so forth). Dissimilar labels in a color group denote statistically significant differences (ie, 5a vs 5b) (P ≤ .05), whereas similar labels in a color group indicate no difference (ie, Sa vs Sa). DAPI, 4',6-diamidino-2-phenylindole. *P ≤ .05, **P ≤ .01, ***P ≤ .001, and ****P ≤ .0001.