Fig. 4: ERRα increases GREM1 expression.

a Schematic diagram showing the positions of putative ERRα binding elements located in the promoter of human GREM1 (http://www.genomatix.de). b Relative mRNA levels of ESRRA in human breast cancer cells. The expression level was quantitated by qPCR analysis. c ChIP assay. The binding of ERRα to the GREM1 promoter was detected by visualisation of the PCR product. Each PCR primer set was designed for the putative ERRα binding elements ‘a’ to ‘d’. d Relative GREM1 promoter activity measured with different GREM1-luciferase (luc) constructs. HEK293 cells were transfected with pGL3-GREM1-luc, which includes all elements, pGL3-GREM1(b + c + d)-luc, which lacks the region ‘a’, or pGL3-GREM1(a)-luc. Luciferase activity was measured and normalised by Renilla activity. pGL3-luciferase construct (pGL3-luc) was used as a negative control. e Effect of XCT790 on GREM1 expression. Cells were treated with XCT790 (1 or 10 μM) for 24 h, and the mRNA levels of KLK3 and GREM1 were quantitated by qPCR analysis. f Effect of ESRRA knockdown on GREM1 expression. Cells were transfected with siCtrl or two siESRRAs (40 nM, each) for 48 h and the lysates were immunoblotted with the indicated antibodies. g Effect of ERRα overexpression on GREM1 expression. Cells were transfected with mock or ERRα in the presence of PGC-1α for 48 h and the mRNA levels of genes were quantified by qPCR analysis. All values in the graphs represent mean ± SD of three independent experiments. Two-sided t test. ***P < 0.001.