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. 2021 Dec 24;26(3):800–812. doi: 10.1111/jcmm.17125

FIGURE 2.

FIGURE 2

Androgen‐induced ZFHX3 transcription upregulation involves AR’s binding to ZFHX3 promoter in PCa cells. (A) Schematic of ZFHX3 promoter region from −2954 to +597 relative to its transcriptional initiation site. Rectangles indicate the 6 potential androgen‐responsive elements (ARE1‐ARE6), and horizontal arrows indicate PCR primers’ locations for ChIP‐PCR (P1F‐P11R). Red and green arrows correspond to R1881‐enhanced and ‐attenuated, respectively, AR‐bound regions revealed by ChIP‐PCR. (B) Relative luciferase activity of the ZFHX3 promoter‐reporter plasmid (pZFHX3‐Luc) in C4‐2B cells undergone hormone depletion for 24 h followed by R1881 treatment for 24 h at 1, 10 and 100 nM. (C) Detection of AR‐bound ZFHX3 promoter DNA in C4‐2B cells using ChIP‐PCR. Cells were pre‐cultured in a hormone‐free medium for 24 h and then treated with R1881 at 1 nM for 6 h before harvesting for ChIP. (D) Confirmation of increased AR binding to ARE1 (P1) and decreased binding to ARE6 (P10) of ZFHX3 after R1881 treatment in C4‐2B cells with extended hormone depletion (72 h) followed by R1881 treatment at 1 nM for 12 h. (E, F) Detection of AR and ZFHX3 in the nucleus and the cytoplasm by Western blotting in LNCaP (E) and C4‐2B (F) cells cultured in complete medium with 10% FBS or in hormone‐free medium (phenol red‐free medium containing 5% charcoal‐stripped FBS, CSS) for 24 or 72 h. Lamin B1 and α‐tubulin serve as nuclear and cytoplasmic markers, respectively. (G) Comparison of conserved AR binding sequence (top), as defined in the JASPAR database, to the predicted 6 ARE sequences in ZFHX3 (middle). Predicted AR binding scores are shown at the right. Mutants for the first ARE (mARE1) and the sixth ARE (mARE6), with mutated conserved nucleotides in blue, are shown at the bottom. (H) Relative luciferase activities of pZFHX3‐Luc, pZFHX3‐Luc‐mARE1, pZFHX3‐Luc‐mARE6 and pZFHX3‐Luc‐mARE(1,6) in C4‐2B cells with 24 h hormone depletion and subsequent 24 h R1881 treatment at 1, 10 and 100 nM. C, cytoplasmic; N, nuclear; ns, not significant; *, p < 0.05; ***, p < 0.001