Fig. 2: ANKRD1 is under direct negative control of AR.

a Left: AR-binding peaks (BEDGRAPH) to the ANKRD1 gene in human primary foreskin fibroblasts (HFF = SG1) by ChIPmentation sequencing. Shown is the IGV representation of 13 KB region encompassing the ANKRD1 gene. Right: RPL10 gene was used as negative control for AR binding, no binding peaks were detected. b Validation of AR binding peaks shown in a in three HDF strains to the ANKRD1 promoter versus a negative site (exon 8 of ANKRD1) by ChIPmentation RT-qPCR. AR binding was quantified relative to non-immune IgGs via qPCR amplification. n(strains) = 3, two-way ANOVA. c Global prediction of transcription factor binding using the Cistrome DB toolkit (http://dbtoolkit.cistrome.org/). Regulatory potential (RP) scores for indicated transcription factors are represented with box plots, with boxes showing the interquartile range, center line representing the median value; minimum and maximum values delineate the range of data points. n(total ChIP datasets analyzed) = 200, top 6 TF are shown. Each point represents a separate ChIP-seq dataset. d ChIP-seq data of transcription factors showing the highest overlap ratio in the genomic region bound by AR (Site 1) using the CistromeDB toolkit. AR and other transcription factors are represented using box plots, with boxes showing the interquartile range, center line representing the median value; minimum and maximum values delineate the range of data points. n(total ChIP datasets analyzed) = 83, top 6 TF are shown. Each point represents a separate ChIP-seq dataset. e RT-qPCR analysis of different HFF strains with or without AR silencing using two different shRNAs (shAR#1, shAR#2) versus shRNA control (shCTRL). Fold change (FC) relative to shCTRL, mRNA normalized to RPLP0. n(strains) = 5. Two-way Anova with Dunnett’s multiple comparison’s correction. f WB for ANKRD1 and AR in HDFs with AR silencing (shAR#1 and shAR#2) compared to control HDFs (shCTRL). Anti-GAPDH was used as a loading control. n(strains) = 3. g RT-qPCR analysis of UT-155-treated HDFs (1 µM, 48 h) compared to DMSO-treated HDFs; expressed as relative FC compared to DMSO. mRNA levels are normalized to RPLP0. n(biological replicates) = 5, mean ± SD, unpaired t-test. h IF of ANKRD1 (green), DAPI (blue) in UT-155-treated HDFs (1 µM, 48 h) compared to DMSO-treated HDFs. n(fields) = 13 (DMSO), 9 (UT-155), mean ± SD, unpaired two-tailed t-test. Scale bar: 20 µm. Data points indicate the average number of cells/fields from three independent experiments. i WB of ANKRD1 and TUBULIN in patient-derived JQ1-treated CAFs (0.5 μM, 48 h) versus DMSO treatment. n(strains)=3.