Fig. 3.

Effect of SNP-dependent regulation of CALML3 and ERα on the expression of ZNF423. a, b Expression of ZNF423 and BRCA1 determined by qRT-PCR after knocking down CALML3 in ERα + ZR75-1 breast cancer cells with ZNF423 WT or variant genotypes. mRNA expression levels shown as the mean of three independent experiments (± SEM), comparisons made by two-tailed Student’s t test. Protein levels were determined by western blot analysis. c Schematic representation shows the role of CALML3 in the SNP-dependent effect of the ER dimer. d, e ChIP-qPCR assays performed using CALML3 antibody after knocking down ERα (d) or vice versa (e) in CRISPR-ZR75-1 cells (WT) and ZR75-1 cells (variant) treated with E2 ± 4-OH-TAM/raloxifen. Primer locations indicated in (c). Enrichment of DNA fragments (three independent experiments, mean ± SEM) between different treatments was compared by two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. 4-OH-TAM 4-hydroxytamoxifene, E2 estradiol, ERα estrogen receptor alpha, ERE estrogen response element, Ral raloxifene, V variant, WT wild type