Fig. 4

rs1821848 modulates NR2C2 binding at the ARID3B promoter (A) Binding affinities between oligos containing the A or G allele of rs1821848 and nuclear extracts from HEPM and HEK293 cells were analyzed via electrophoretic mobility shift assay (EMSA). (B and C) RT-qPCR analysis of ARID3B expression after plasmid overexpression and siRNA-mediated knockdown of NR2C2. (D) NR2C2 binding surrounding rs1821848 was observed in HeLa-s3 and K562 cells from the Cistrome database. (E and F) NR2C2 binds to the region flanking rs1821848, as determined by ChIP-qPCR (E) and DNA electropherogram (F). (G and H) Effect of NR2C2 overexpression (G) or NR2C2 knockdown (H) on the relative luciferase activity of constructs containing the A or G allele of rs1821848. (I) Binding affinities between biotin-labeled oligonucleotides containing the A or G allele of rs1821848 and NR2C2 were analyzed by EMSA. Nuclear extracts were obtained from HEPM cells transfected with either an empty vector or pcDNA3.1-NR2C2. (J) Sanger sequencing showed that rs1821848 was homozygous in HEPM and HEK293 cells. (K) Full-length ARID3B expression constructs containing either the A or G allele of rs1821848 were transfected into ARID3B-knockout cells. (L) ChIP-ASE analysis showed significantly stronger NR2C2 binding to the rs1821848-G allele compared to the A allele. Data are presented as mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns, not significant.