Figure 1
Up-regulation of NF-κB pathway protects NB cells from tepotecan-mediated growth inhibition. (A) Quantitative RT-PCR was performed on the 5 enhancer genes (Birc4, RIPK1, NfKB1, TNFRSF8 and TNFRSF25) to confirm target gene knockdown. Each gene expression is normalized with GAPDH and compared to siRNA control. Error bar represents the standard deviation of triplicate experiment. (B) GSEA analysis was performed on the ranked genes according to log2 ratio of gene expression between topotecan-treated and control SK-N-AS cells. The NF-κB target genes are significantly enriched in SK-N-AS cells treated with topotecan at 1 μM (p < 0.0001). The green curve shows the running sum of enrichment score (ES) for the ranked genes. Black vertical lines indicate gene hits in the NF-κB target gene set. The red vertical line marks the leading edge subset genes, and the NF-κB target genes within the leading edge subset genes are shown in their gene symbols. (C) Immunoblots showed activation of the NF-κB pathway by topotecan (5 μM) through phosphorylation and degradation of total IκB-α protein; and phosphorylation of p65/RelA in a time dependent manner. (D) Knockdown of NFKB1 potentiated the inhibitory effect of topotecan on cell growth when compared to topotecan alone (p = 0.0086), or NFKB1 siRNA alone (p = 0.0045) on a real-time cell electronic sensing system. (E) Immunoblots confirmed knockdown of NFKB1 protein (p105/p50 complex) of SK-N-AS cells transfected with NFKB1 siRNA for 72 hrs compared to scramble siRNA