Figure 4. MYCN inhibits CD9 expression.

A.-B., CD9 expression was assessed on the mRNA (qRT-PCR; mean ± SD, n = 3) and protein level (western blots) in the synthetic MYCN-inducible SH-EP Tet-21/N cell model with/without MYCN induction in time-course A. and in BE(2)-C at designated time points after MYCN depletion B. CD9 protein expression in SH-EP Tet-21/N was below detection. GAPDH served as loading control. C., ChIP-qPCR showing an enrichment of CD9 promoter DNA associated with MYCN. BE(2)-C lysates were immunoprecipitated with antibodies against MYCN or IgG, as negative control, and 0.1% of the input served for normalization (mean ± SD, n = 3). D., CD9 expression was assessed on the mRNA level (qRT-PCR; mean ± SD, n = 3) in BE(2)-C 96h after GRHL1 knockdown using a small interfering RNA (si-1) and 72h after MYCN depletion using a short hairpin RNA (shRNA) directed against MYCN. Controls were transfected with respective negative control siRNA and empty vector. E., the expression of Cd9 during neuroblastoma progression from tumor-prone ganglia to tumors in transgenic mice (n = 4; full line) and in comparison to Cd9 expression in wild-type ganglia (n = 4; dashed line). Linear regression analysis; P = 4.7×10⁻¹¹; delta slope = −1.1. ^*P < 0.05, ^**P ≤ 0.01.