Figure 3
From: RNA-binding motif protein 47 inhibits Nrf2 activity to suppress tumor growth in lung adenocarcinoma
RNA-seq and RIP-seq analyses reveal that knockdown of RBM47 elevates Nrf2 activity. (a) A549-Luc cells were infected with lentiviral vector encoding control shRNA (shCT) or shRNAs against RBM47 (shRBM47#1 and #2). RBM47 expression was examined in A549-Luc-shCT cells and A549-Luc-shRBM47 cells by immunoblot analysis. (b) Enrichment of Nrf2 targets in the list of genes that were upregulated by knockdown of RBM47. Gene expression data obtained by RNA-seq in A549-Luc-shCT cells and A549-Luc-shRBM47#1 cells were used for gene set enrichment analysis. A gene set NFE2L2.V2 of c6 MSigDB oncogenic signature (v4.0) consisting of the direct gene targets of Nrf2 was identified as the second most enriched phenotype of the analysis; its enrichment plot is shown. Normalized enrichment score (NES)=1.854. Expression data of the genes whose expression levels exceeded 10 fragments per kilobases of exon per million sequence read (FPKM) in any of the samples were used for the analysis. (c) qRT–PCR analysis of the identified Nrf2 target genes regulated by RBM47. (d) RIP-seq data of Nrf2 and its regulatory genes. RIP-seq was performed in A549 cells using anti-RBM47 or normal rabbit IgG (rIgG) and SNRNP70 as a control. RBM47_rep1 and RBM47_rep2 represent biological replicates of anti-RBM47 RIP. Quantified FPKM values of RBM47 RIP samples were normalized against rIgG-RIP-seq FPKM. (e) Immunoblotting of KEAP1 and CUL3 proteins in A549-Luc cells expressing shCT or shRBM47s. Protein expression was quantified using Image J and normalized to that of lane 1 and is indicated below each panel. (f) qRT–PCR analysis of KEAP1 and CUL3 expression in cells after RBM47 knockdown. (g) A quantitative ChIP–PCR analysis of Nrf2 binding at its target-binding regions in A549 cells. HBB: control genomic region. (h) qChIP–PCR analysis was performed in H441 cells after knockdown of RBM47 expression by siRNAs (siRBM47#1 and siRBM47#2). siCT: control siRNA. In (g) and (h), averages and standard deviations of the three technical replicates were shown for each condition. (i) Expression of small Maf family and CDKN1A was determined by RNA-seq. Error bars: 95% confidence interval calculated by Cuffdiff.
