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Fig. 2
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Figure Caption
Fig. 2. Transient in vitro RRM2 knockdown in neuroblastoma cells results in an increased DNA damage and p53 pathway response, supporting its putative dependency role in neuroblastoma.
(A) Transient RRM2 knockdown in IMR-32 and CLB-GA neuroblastoma cells using two different RRM2-targeting siRNAs (denoted as si61 and si62) significantly down-regulates the expression of RRM2 and up-regulates the expression of the p53 target gene CDKN1A and the p53-inducible RRM2B gene, as shown by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis. ns, not significant. (B) Immunblotting confirms that RRM2-targeting siRNA transfection in IMR-32 and CLB-GA cells strongly down-regulates RRM2 protein levels accompanied by DNA damage induction (increased pRPA32 and yH2AX signal) and checkpoint activation (increased pCHK1 levels) (see quantification in fig. S2). (C) IncuCyte live cell imaging analysis shows a strong reduced confluency upon siRRM2_61 transfection and, to a lesser extent, with siRRM2_62 in IMR-32 and CLB-GA cells. (D) GSEA following RNA sequencing (RNA-seq)–based transcriptome profiling of IMR-32 and CLB-GA cells transfected with RRM2-targeting siRNAs indicates a significant reduced expression of MYC targets and G2-M phase markers (top) and up-regulation of p53 target genes (bottom). FDR, false discovery rate; NES, normalized enrichment score.
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