a, Immunoblot showing expression of endogenous and V5-tagged exogenous FOXA1 proteins in doxycycline (dox)-inducible 22RV1 cells transfected with distinct UTR-specific FOXA1-targeting siRNAs (no. 3–5) or a non-targeting control siRNA (siNC). These results represent two independent experiments. IncuCyte growth curves of 22RV1 cells overexpressing empty vector (control), wild-type or mutant FOXA1 variants upon treatment with UTR-specific FOXA1-targeting siRNAs (n = 5 biological replicates). Mean ± s.e.m. is shown. b, Immunoblots confirming stable overexpression of the wild-type AR protein in HEK293 and PC3 cells. c, d, Co-immunoprecipitation assay of indicated recombinant FOXA1 variants using a V5-tag antibody in HEK293 (c) and PC3 (d) cells stably overexpressing the AR protein (referred to as HEK293-AR and PC3-AR cells). eGFP is a negative control. FOXA1-FL, full-length wild-type FOXA1. del168 and del358 are truncated FOXA1 variants with only the first 168 amino acids (that is, before the FKHD) or 358 amino acids of the FOXA1 protein. H247Q and R261G are missense class-1 mutant variants. e, Immunoblots confirming comparable expression of AR and recombinant FOXA1 variants in AR reporter assay-matched HEK293 lysates. Immunoblots show representative results from 2 or 3 independent experiments and class-1 and class-2 mutants serve as biological replicates. For all gel source data (a, b–e), see Supplementary Fig. 1. f, AR dual-luciferase reporter assays with transient overexpression of indicated FOXA1 variants in HEK293-AR cells with or without DHT stimulation and enzalutamide treatment (n = 3 biological replicates per group). Mean ± s.e.m. is shown (two-way ANOVA and Tukey’s test). g, Genes differentially expressed in class-1 tumours from patients (n = 38) compared to FOXA1 wild-type tumours (see Methods). The most significant genes are shown in red and labelled (limma two-sided test). h, Differential expression of cancer-hallmark signature genes in class-1 mutant prostate-cancer tumours (GSEA statistical test). i, Localized, primary prostate cancer gene signature showing concordance between class-1 tumour and primary prostate cancer genes. j, BART prediction of specific transcription factors mediating observed transcriptional changes. The significant and strong (z-score) mediators of transcriptional responses in class-1 tumours are labelled (BART, Wilcoxon rank-sum test). k, mRNA expression (RNA-seq) of class-1 signature genes in LNCaP and VCaP cells either starved for androgen (no DHT) or stimulated with DHT (10 nM). RNA-seq from two distinct prostate cancer cell lines is shown. l, Representative FOXA1 and AR ChIP–seq normalized signal tracks at the WNT7B or CASP2 gene loci in LNCaP and C42B cells. ChIP–seq assays were carried out in two distinct prostate cancer cell lines with similar results. m, Growth curves (IncuCyte) of 22RV1 cells overexpressing distinct FOXA1 variants in complete, androgen-supplemented growth medium (n = 2 biological replicates). Mean ± s.e.m. is shown. n, Per cent viable 22RV1 stable cells, overexpressing either empty vector, wild-type or mutant FOXA1 variants upon treatment with enzalutamide (20 μM for 6 days; n = 4 biological replicates). Mean ± s.e.m. is shown. P values in m and n were calculated using two-way ANOVA and Tukey’s test. o, p, mRNA expression (RNA-seq) of labelled basal and luminal transcription factors or canonical markers in FOXA1 wild-type, class-1 or class-2 mutant tumours in primary prostate cancer (total n = 500; two-way ANOVA). q, Extent of AR and neuroendocrine (NE) pathway activation in FOXA1 wild-type, class-1 or class-2 mutant cases from both primary (n = 500) and metastatic (n = 370) prostate cancer. Both AR and NE scores were calculated using established gene signatures (see Methods). Left, two-sided t-test; right, two-way ANOVA. For all box plots, centre shows median, box marks quartiles 1–3 and whiskers span quartiles 1–3 ± 1.5 × IQR. Source Data