Extended Data Figure 8. Functional association of FOXA1 and TLE3 in prostate cancer.

a) mRNA (qPCR) and protein (immunoblot) expression of TLE3 in a panel of PCa cells. Mean ± s.e.m and individual data points are shown. b) Left, mRNA expression of FOXA1 and TLE3 in LNCaP and VCaP cells treated with siRNAs targeting either FOXA1 or AR (n=3 technical replicates). Two FOXA1 WT PCa cells serve as biological replicates. Mean ± s.e.m and individual data points are shown. Right, protein expression of FOXA1 and TLE3 in matched LNCaP lysates. c) FOXA1 N-terminal ChIP-seq normalized signal tracks from LNCaP, C42B and LAPC4 PCa cells at the TLE3 locus. Each cell line serves as a biological replicate. d) Overlap of the union WT FOXA1 and TLE3 binding sites from LNCaP, C42B and 22RV1 PCa cells (n=1 for each), and top de novo motifs discovered (HOMER: hypergeometric test) in the TLE3 cistrome. e) Co-immunoprecipitation assays of labelled recombinant FOXA1 WT, class1, or class2 variants using a V5-tag antibody in HEK293 cells overexpressing the TLE3 protein. V5-tagged GFP protein was used as a negative control. These results were reproducible in two independent experiments and distinct class1 and class2 mutant serve as biological replicates. f) Overlap of union TLE3 cistromes from isogenic WT (n=2 biological replicates) or heterozygous class2-mutant (n=2 biological replicates) 22RV1 CRISPR clones. g) ChIP peak profile plots from TLE3 ChIP-seq in isogenic FOXA1 WT or class2-mutant 22RV1 clones (n=2 biological replicates each). h) Representative TLE3 and FOXA1 ChIP-seq read signal tracks from independent 22RV1 CRISPR clones with or without endogenous FOXA1 class2 mutation (n=2 biological replicates each). i) Gene set enrichment analyses showing significant enrichment of (left) WNT and (right) EMT pathway genes in 22RV1 cells treated with TLE3-targeting siRNAs (n=2 biological replicates for each treatment; GSEA enrichment test). j) Left, mRNA (RNA-seq) expression of direct WNT target genes in 22RV1 upon siRNA-mediated knockdown of TLE3 (n=2 biological replicates). Right, Immunoblot showing LEF1 up-regulation upon TLE3 knockdown in 22RV1 PCa cells with and without androgen starvation (representative of two independent experiments). For gel source data (a-d,j), see Supplementary Figure 1. k) Gene enrichment plots showing significant enrichment of class2 up-regulated genes upon TLE3 knockdown in 22RV1 cells (n=2 biological replicates for each treatment; GSEA enrichment test).