Generation and characterization of ESR1 wild-type and D538G mutant models. (A) CRISPR-mediated epitope tagging strategy was used to generate heterozygous FLAG-tagged wild-type ESR1 and D538G mutant ESR1 Ishikawa cell lines. (B) Immunoblotting for FLAG and ESR1 in Ishikawa parental cells, two heterozygous FLAG-tagged wild-type and three heterozygous FLAG-tagged D538G mutant cell lines show protein expression of epitope-tagged ESR1 and total ESR1. (C) The ESR1 wild-type and mutant allele expression frequencies based on RNA-seq data is shown for each D538G clonal cell line. (D) Estrogen response element (ERE) reporter activity as measured by luciferase activity was assayed in DMSO- and E2-induced conditions. Experiments were performed in triplicate, and the average luciferase activity for two wild-type and three D538G mutant clones is shown. (***) P = 0.0002; (****) P < 0.0001; error bars represent SEM.
