Gene expression study on ENSc stimulated with Wnt3a and LPS. (A) The anti-inflammatory effect was demonstrated to be exerted by Wnt3a using qPCR analysis and ENS cells treated with Wnt3a (20 ng/mL) and LPS (5 μg/mL). The analysis was focused on the expression of β-catenin target genes (AXIN2, CMYC, and CJUN), membrane receptors (TLR4 and FZD9), Wnt3a ligand, and pro- and anti-inflammatory target genes (IL1B, IL6, TNFa, and IL10). The amount of gene products was calculated using linear regression analysis from standard curves, demonstrating the amplification efficiencies ranging from 90% to 100%. Data were reported as a fold increase of gene expression that is defined as the cDNA ratio between target gene and reference gene (HPRT) normalized to untreated sample. Statistical significance was calculated using Student’s t-test, comparing to untreated cells: p value ≤0.05*, p value ≤0.01**; samples compared to LPS-treated cells: p value ≤0.01 (two black triangles). (B) To detect the gene expression of typical ENS growth factors (GDNF, EGF, BFGF, NGF, and LIF), RT-PCR was performed using Qiagen One Step RT-PCR Kit on samples untreated (T0) and stimulated with Wnt3a (20 ng/mL) and LPS (5 μg/mL) for 1 (T1) and 7 (T7) days. RT-PCR products were electrophoresed on 2% agarose gel and stained by GelRed™. HPRT was used as housekeeping gene.