Figure 2.

High RSPO3 expression in lung AD depends on Keap1 deficiency and RSPO3 promoter demethylation. (a) A heat map depicting the expression levels of RSPO3 and three NRF2-regulated genes vs. mutation and expression status of Keap1 in TCGA’s LUAD cohort. (b) Kaplan-Meier survival analysis of patients with tumors of Keap1 deficiency and high RSPO3 expression vs. those with tumors of only Keap1 deficiency and the rest of the cohort. (c) Representative WB of A549 cells stably expressing pLVX-GFP or pLVX-GFP-Keap1 with anti-GFP antibody (left panel), anti-Keap1 antibody (mid-panel) and anti-NRF-2 antibody (right panel). eKEap1: endogenous Keap1. The experiment was repeated twice. (d) RT-qPCR results of AKR1C2 and RSPO3 in GFP vector and GFP-Keap1-expressing cells. Expression data were normalized by 18S RNA and error bars are S.E.M. of three replicate experiments. (e) Representative ChIP-PCR results of three putative NRF2 binding sites located at -6824, -6043, and -5620 bp upstream of the TSS (transcription start site) of RSPO3. The antibodies used in the lanes are: 1 = anti-PolII, 2 = control IgG, 3 = anti-NRF2. Lanes 4 (no DNA) and 5 (input chromatin) are negative and positive controls, respectively. The experiments were repeated twice. (f) Comparison of methylation beta value of 22 CpG’s across the RSPO3 promoter region in RSPO3-high vs. RSPO3-norm tumors. (g) RT-qPCR results of RSPO3 expression in H2009 cells treated with vehicle, sulforaphane (SF, 10 μM) and azacytidine (AZ, 10 μM), and SF and AZ simultaneously (SF + AZ). Expression data were normalized by 18S RNA and error bars are S.E.M. of three replicate experiments.