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. 2023 May 4;14(5):304. doi: 10.1038/s41419-023-05836-7

Fig. 2. CHRM4 is downregulated by the AR in prostate cancer cells.

Fig. 2

A ChIP-sequencing analysis of the detected DNA-binding sites for the AR of the CHRM4 gene in cells in response to 0.5 or 4 h of AR-ligand R1881 treatment labeled as black boxes in the tracks. ChIP-sequencing data were downloaded from the Gene Expression Omnibus (GEO) (GSE84432) and analyzed by Genome Brower (Genomics Institute). B Schematic of the predicted AR resonse elements (AREs) and an introduced single- or double-binding site mutant in regulatory sequence reporter constructs of human CHRM4 (GRCh38:11). ChIP assay showing binding of the AR and acetyl-H3 to predicted AREs of the CHRM4 gene regulatory sequence following treatment of C4-2 cells with 10 nM dihydrotestosterone (DHT) (C) or 20 μM MDV3100 (D) for 24 h. Sheared chromatin from nuclear extracts was precipitated with antibodies to the AR and acetyl-H3, and predictive primers (B, black arrows) were used to quantify the precipitated DNA by a qPCR. Enrichment of each protein to each site is given as a percentage of the total input and then normalized to IgG. * vs. the vehicle (Veh) (C) or DMSO (D), by a one-way ANOVA. E, F ChIP assay showing binding of the AR and acetyl-H3 to predicted AREs of the CHRM4 gene regulatory sequence in PC3 cells following stable transfection with an empty vector (EV) or AR cDNA vector (E) or in C4-2 cells with a non-targeting control (NC) or AR siRNA transfection (F). * vs. the EV (E) or NC (F), by a one-way ANOVA. G Relative CHRM4 and AR mRNA levels of C4-2 cells transfected with the NC or AR siRNA, measured by an RT-qPCR analysis, * vs. the NC. H, I Relative mean florescence intensity (MFI) of the GFP reporter gene containing a wild-type (WT)- or mutant (M)-ARE from the CHRM4 regulatory sequence in C4-2 cells following treatment with 10 nM DHT (H) or 20 μM MDV3100 (I) for 48 h. * vs. WT; # vs. the vehicle (Veh) (H) or DMSO (I), by a two-way ANOVA. J, K Relative MFI of the GFP reporter gene containing a WT- or M-ARE from the CHRM4 regulatory sequence in PC3 cells following stable transfection with the EV or AR cDNA vector (J) or in C4-2 cells following NC or AR siRNA transfection (K). * vs. the EV (J) or NC (K), by a two-way ANOVA. Quantification of the ChIP assay, relative MFI values, and mRNA levels are presented as the mean ± SEM from three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001.