Fig. 4: NKX2-1 is induced by FOXA2 and required for FOXA2-driven NET.

a, Genome browser view of NKX2-1 mRNA (top row), and FOXA2, H3K4me1, H3K27ac ChIP–seq signal around the NKX2-1 gene in time-course LNCaP+FOXA2 cells. The promoter is highlighted in light blue, and enhancers are highlighted in yellow. b, WB of NEPC cell lines (LuNE, NCI-H660) and organoids (LuCaP145.2) with control or FOXA2 KD. Data shown are from one of two (n = 2) independent experiments. LuNE cells—stable LNCaP+FOXA2 cells. c, FOXA2 Co-IP showing its interaction with NKX2-1 protein in NEPC cell line NCI-H660. Data shown are from one of three (n = 3) independent experiments. d, Heatmap showing AR, NKX2-1 and FOXA2 occupancy at previously reported¹¹ NE-CREs (n = 14,985) and Ad-CREs (n = 4,338) in LNCaP or NEPC models (NCI-H660, LuCaP93, LuCap145.1, LuCaP145.2). Scale bar—enrichment intensity. e, Heatmap showing active (H3K27ac) enhancer (H3K4me1) and promoter (H3K4me3) marks at NKX2-1-only, FOXA2-only and shared binding sites in LuCaP145.1. Scale bar—enrichment intensity. f, APA plots of FOXA2- and NKX2-1-anchored E–P loops identified in D28 in time-course LNCaP+FOXA2 Hi-C samples. The APA score is annotated within each plot. Scale bars indicate log₂ of observed over expected enrichment. g, NKX2-1 KD abolished FOXA2-induced NET. LNCaP cells were co-infected with FOXA2 virus along with either sgNC or sgNKX2-1 virus. The infected cells were collected at the indicated time points for WB analyses of luminal and NE markers. The red star indicates a nonspecific band. Data shown are from one of three (n = 3) independent experiments. h, Heatmap showing FOXA2, NKX2-1, H3K27ac and H3K4me1 ChIP–seq around (±2 kb) the six ATAC–seq peak clusters in LNCaP+FOXA2 cells, with or without NKX2-1 KD, at indicated time points. Peaks within each cluster were separately sorted by FOXA2 ChIP–seq intensity. Scale bar—enrichment intensity.