Fig. 3: BRAFi treatment of melanoma cells increases AR expression in melanoma cells through an AR-positive feedback loop.

a Tornado plots of AR binding sites, as determined by ChIP-seq analysis, 5,000 bp upstream and downstream of TSS, in A375 cells treated for 48 hrs with DAB versus vehicle (DMSO). The heatmap depicts the ChIP-seq signal at lower (white) and higher (red) peak intensity. Quantification of lost and gained AR binding peaks in DAB-treated versus control cells. b Functionally relevant gene ontology (GO) families of the genes significantly upregulated by BRAFi treatment in three melanoma lines and bound by AR (by ChIP seq) in DAB-treated A375 cells. The p-value (-log₁₀) is indicated on the x-axis, unpaired two-tailed t-test. The differentially expressed genes are provided in Supplementary Data 2. c Top target genes bound by AR in DAB-treated A375 cells, as identified by ChIP-seq, using the Cistrome DB toolkit (http://dbtoolkit.cistrome.org/). RP = Regulatory potential. d Illustration of AR-binding peaks within AR locus, determined by ChIP-seq and displayed using the integrative genomic viewer software (IGV), in DAB-treated A375 cells (deep purple) versus control (cyan). H3K27ac peaks (light purple), map histone modifications overlapping with AR binding regions, were derived from⁵⁴. e Top transcription factors with predicted overlapping binding with AR to the AR locus using the Cistrome DB toolkit (http://dbtoolkit.cistrome.org/). Each dot represents the average overlap ratio derived from individual studies. y-axis: names of transcription factors; x-axis: overlap ratio values. f RT-qPCR analysis of AR expression in A375 melanoma cells treated with inhibitors of the indicated molecules/pathways for 48 h. AR expression (arbitrary units) relative to untreated controls, after RPLP0 normalization. Mean ± SD, n (biological replicates)=2. g AR IF in A375 melanoma cells treated with inhibitors of the indicated molecules/pathways (48 h). Representative images and quantification of AR nuclear signal (arbitrary units). Mean ± SD, n (cells/sample)>200, unpaired, two-tailed t-test, ****p < 0.0001. Color scale: red, DAPI; cyan, AR. Scale bar: 20 μm.