Fig. 3.

Silencing TFAP4 selectively inhibits growth of MYCN-amplified neuroblastoma cell lines. a Schema of cell growth competition assay. SHEP21N cells infected with either shTFAP4-GFP or an empty vector control pGIPZ-GFP were mixed with equal number of uninfected SHEP21N cells. The infected cells to WT cells ratio is measured by FACS. b TFAP4 gene expression (qRT-PCR) in SHEP21N-pGIPZ control cells and SHEP21N expressing shTFAP4. c %GFP ratio measured at day 4, 7, 10, and 14. Experiments were performed in triplicate. Mean ± std dev. d % of neuroblastoma cell survival 96 h after siRNA-mediated downregulation of TFAP4. Neuroblastoma cell lines were transfected with four different constructs of siRNA against TFAP4. The experiments were performed in quadruplicate. Mean ± std dev. *, P < 0.05; **, P < 0.01; ***, P < 0.001. e Proliferation assays showing neuroblastoma cell growth after silencing TFAP4 by doxycycline-inducible shRNAs. Neuroblastoma cell lines were infected with two different dox-inducible shRNAs against TFAP4 as well as the empty vector control pTRIPZ. shRNA was induced by 1 μg/ml doxycycline at day 0. Cells were counted on day 0, 2, 4, and 6. Experiments were performed in triplicate. Mean ± std dev. *, P < 0.05; TFAP4 silencing was confirmed by western blot. f Colony formation assay. Neuroblastoma cells infected with doxycycline-inducible shRNAs were plated in semisolid agar media for 21 days and stained with MTT. Experiments were done in triplicate. Mean ± std dev. *, P < 0.05; **, P < 0.01