Figure 1
From: High-throughput cell-based screening reveals a role for ZNF131 as a repressor of ERalpha signaling
High-throughput screening of ERE reporter activity inhibitors in HeLa cells. (A) Candidate genes, cloned into the mammalian expression vector pcDNA3.1 (pcDB), were tested for their ability to inhibit the response, to 17β-estradiol (10 nM), of and ERE-luciferase reporter. Cells cultured in 96-well plates were co-transfected in parallel with plasmids of pGL4-ERE-LUC, pRL-TK, ERα and candidate gene or pcDB. Results were corrected for transfection efficiency against Renilla luciferase activity. Activity in the presence of the empty pcDB vector alone was given an arbitrary value of one. BRCA1, the positive control, yielded a luciferase activity of 0.6 (red arrow). 8 clones in the first round of screening exhibited chemiluminescence values lower than this (red spots). (B) 4 of the 8 candidate genes (TRAF3IP3, ING4, ZNF131, and PHF7) passed a repeat triplicate screening, with ZNF131 identified as the most potent ERα transcription repressor. (C) ZNF131 repressed ERα transcriptional activity in a dose-dependent manner. HeLa cells were transiently transfected with pGL4-ERE-LUC (40 ng), pRL-TK (5 ng), ERα (0.1 ng) and increasing amounts (0, 10, 25, 100, 250, and 1000 ng) of ZNF131 expression plasmids. Cells were treated with 10 nM 17β-estradiol (E2). Data in (B and C) are presented as mean ± SD of three independent experiments. (*, significant difference from control values; P < 0.05)