NANOG knockdown decreases WNT3A-induced HUVEC proliferation. (A) Timeline of knockdown, FLK1 cDNA transfection (rescue), and the BrdU assay. (B) Quantification of BrdU incorporation in control HUVECs and HUVECs treated with control siRNA, NANOG siRNA, or NANOG siRNA + FLK1 cDNA (siRNAs used for this experiments were chemically synthesized) (knockdowns were performed in OPTI-MEM media + WNT3A). The cells were incubated with BrdU (1.0 μg/mL) for 16 hours in media containing WNT3A (without serum), then fixed, stained, and quantified. For quantification, ≥ 10 random fields were selected from each coverslip and examined under ×200 magnification. The data represent the mean ± SEM; n = 6-10 from 5-7 independent experiments; *P < .05 and **P < .01 compared with control. (C-F) Representative images of the cell proliferation assays. White arrows indicate BrdU-positive cells. (G) The efficiency of NANOG knockdown and FLK1 cDNA reexpression was determined by WB analysis with the indicated antibodies. The NANOG protein appeared as a 45- to 50-kDa polypeptide. Arrowheads indicate 230-, 200-, and 150-kDa anti-FLK1 antibody immunoreactive bands. Anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to determine equal protein loading. Experiments were performed ≥ 3 times.