NANOG-knockdown disrupts formation of branching point structures. (A) Timeline of experiment. (B) Quantification of branching points. To deplete endogenous NANOG, human LMVECs at 50% density (2 × 106) were either left untreated or infected with control shRNA or NANOG shRNA (retrovirus) for 16 hours (in presence of serum and growth factors). For rescue experiment, FLK1-cDNA plasmid (1.5 μg/2 × 106 cells) was included along with NANOG-shRNA. Cells were detached with 2mM ethylenediaminetetraacetic acid, pH 7.4, washed with PBS, then plated onto 3-dimensional type I collagen matrix + WNT3A (50 ng/mL) (no serum and growth factors added from this time point). After 36 hours, the number of branching points were counted. Data are expressed as the percentage of branching points; n = 5; *P < .05 compared with control or as indicated. (C-F) Representative images of branching point structures. Experiments were repeated 3 times with the use of triplicate wells. Magnification, ×200. Scale bar, 200μm. Arrows indicate branching points. (G) Whole-cell lysates were subjected to WB analyses with the antibodies indicated. All blots shown are representative of 3 independent experiments.
