Extended Data Fig. 2: Transcriptional suppression of AR on GREM1 depends on a high androgen concentration.
(a-i) Immunoblotting and q-PCR analysis of GREM1 expression, and GREM1 promoter-driven luciferase assay in AR overexpressed LNCaP (a, b, g) or LAPC4 cells (c, d, h) and AR knock-out LNCaP cells (e, f, i) (b, d, n = 4 independently treated cell cultures, f, n = 3 independently treated cell cultures, g, h, i, n = 3 independently transfected replicates). (j, k) The AR gene transcription is upregulated in LNCaP-R and VCAP cells treated with charcoal/dextran-stripped FBS for 3 weeks (VCAP-R) (n = 3 independently treated cell cultures). (l, m) LNCaP or LNCaP-R cells and VCAP or VCAP-R cells were treated with 0, 0.01, 0.1, 1, or 10 nM DHT for 24 hrs. GREM1, KLK3 and OPRK1 mRNA are measured by q-PCR analysis (n = 3 independently treated cell cultures). (n) 0, 0.1 or 10 nM DHT were added to LNCaP cells for 4 hr. Binding of AR to the ARE of GREM1 or KLK3 gene is measured by ChIP q-PCR. (o) LNCaP-R cells were treated with or without 10 nM DHT. The enrichment of AR to GREM1 and KLK3 gene is measured by ChIP q-PCR (n = 3 independently treated cell cultures). (Two-tailed Student’ s t test was used for the statistical analysis. Data are presented as means ± s.e.m.).
