T later identified in the human 59UTR which was reported to be responsible for the decrease of AR promoter activity in androgen-dependent LNCaP cells [32].Figure 4. Comparison of gene expression in LN/TC-AR following induction of TC-AR or treatment with DHT. A Venn diagram of microarray analyses of LN/TC-AR cells treated with 1 nM DHT or different concentrations of doxycycline for 24 hours post serum starvation. Numbers indicate genes that were commonly or uniquely upregulated (q) or downregulated (Q) following the various treatments. B LN/TC-AR cells were treated with Low Dox, High Dox, 1 nM DHT, or GSK -3203591 vehicle in hormone depleted media for 24 hours. Quantitative RT-PCR was performed for the selected genes. Fold induction is relative to 18334597 values obtained in untreated control using identical gene specific primers. doi:10.1371/journal.pone.0049887.gModeling Truncated AR in AD BackgroundFigure 5. Knockdown of RHOB affects cell shape and cell migration following overexpression of TC-AR in LN/TC-AR/shR-RHOB. A LN/TC-AR cells were pre-cultured in hormone-depleted media for 48 hours then treated with Low Dox, High Dox, 1 nM DHT or vehicle for 24 hours. Whole cell lysates were harvested and subjected to western blot analysis. Membranes were probed with a-RHOB or a-actin. B Graphic representation of TC-AR binding sites 100 kb within the region of adjacent to RHOB gene. Arrows above show TC-AR binding sites (+3.8 kb and +47.5 kb downstream of TES). C Androgen-deprived LN/TC-AR/shR-empty and LN/TC-AR/shR-RHOB cells were treated with 1 nM DHT, Low Dox, High Dox or vehicle only. Whole cell lysates were harvested 48-hours post-treatment and subjected to western blot analysis. Membranes were probe with either a-RHOB (top) or a-actin (bottom). D Androgen-deprived LN/TC-AR/shR-RHOB cells were treated with 1 nM DHT, Low Dox, High Dox or vehicle. Images were acquired 48-hours post-treatment. E LN/TC-AR/shR-RHOB cells were pre-cultured in serum free media (SFM) for 24 hours 1676428 then seeded to migration chambers with various treatments in the presence of SFM for an additional 48 hours after which time fluorescence was detected. Fold induction is relative to untreated control. Prior results for LN/TC-AR (from Figure 3B) are included for comparison. F MTT assay of LN/TC-AR/shR-RHOB in hormone depleted media following treatment with 1 nM DHT, Low Dox, High Dox or vehicle as control showing that DHT independent growth previously shown for LN/TC-AR (Figure 2D) is not inhibited by knockdown of RHOB. Bright field images were acquired with an Olympus microscope using 206 I-BRD9 web magnification. doi:10.1371/journal.pone.0049887.gModeling Truncated AR in AD BackgroundMost recently, AR auto-repression was found to be due to the assembly of a FL-AR repressive complex containing lysine-specific demethylase 1 at the intron 2 enhancer [33]. We show here that AR autorepression is not limited to liganded FL-AR, but is effected by TC-AR as well. Unlike the similar ARv567es, co-immunoprecipitation assays revealed no detectable heterodimerization between FL-AR and TC-AR either in a transient transfection assay [2] or in LN/TC-AR (Figure 1E). While this result excludes repression by FL-AR/TC-AR heterodimers, a similar mode of repression by TC-AR homodimers is plausible and warrants further investigation within the context of LN/TC-AR and other truncated AR models. At a minimalistic level, three criteria must be met for a truncated form of AR to be considered a potential surrogate for DHT-bound F.T later identified in the human 59UTR which was reported to be responsible for the decrease of AR promoter activity in androgen-dependent LNCaP cells [32].Figure 4. Comparison of gene expression in LN/TC-AR following induction of TC-AR or treatment with DHT. A Venn diagram of microarray analyses of LN/TC-AR cells treated with 1 nM DHT or different concentrations of doxycycline for 24 hours post serum starvation. Numbers indicate genes that were commonly or uniquely upregulated (q) or downregulated (Q) following the various treatments. B LN/TC-AR cells were treated with Low Dox, High Dox, 1 nM DHT, or vehicle in hormone depleted media for 24 hours. Quantitative RT-PCR was performed for the selected genes. Fold induction is relative to 18334597 values obtained in untreated control using identical gene specific primers. doi:10.1371/journal.pone.0049887.gModeling Truncated AR in AD BackgroundFigure 5. Knockdown of RHOB affects cell shape and cell migration following overexpression of TC-AR in LN/TC-AR/shR-RHOB. A LN/TC-AR cells were pre-cultured in hormone-depleted media for 48 hours then treated with Low Dox, High Dox, 1 nM DHT or vehicle for 24 hours. Whole cell lysates were harvested and subjected to western blot analysis. Membranes were probed with a-RHOB or a-actin. B Graphic representation of TC-AR binding sites 100 kb within the region of adjacent to RHOB gene. Arrows above show TC-AR binding sites (+3.8 kb and +47.5 kb downstream of TES). C Androgen-deprived LN/TC-AR/shR-empty and LN/TC-AR/shR-RHOB cells were treated with 1 nM DHT, Low Dox, High Dox or vehicle only. Whole cell lysates were harvested 48-hours post-treatment and subjected to western blot analysis. Membranes were probe with either a-RHOB (top) or a-actin (bottom). D Androgen-deprived LN/TC-AR/shR-RHOB cells were treated with 1 nM DHT, Low Dox, High Dox or vehicle. Images were acquired 48-hours post-treatment. E LN/TC-AR/shR-RHOB cells were pre-cultured in serum free media (SFM) for 24 hours 1676428 then seeded to migration chambers with various treatments in the presence of SFM for an additional 48 hours after which time fluorescence was detected. Fold induction is relative to untreated control. Prior results for LN/TC-AR (from Figure 3B) are included for comparison. F MTT assay of LN/TC-AR/shR-RHOB in hormone depleted media following treatment with 1 nM DHT, Low Dox, High Dox or vehicle as control showing that DHT independent growth previously shown for LN/TC-AR (Figure 2D) is not inhibited by knockdown of RHOB. Bright field images were acquired with an Olympus microscope using 206 magnification. doi:10.1371/journal.pone.0049887.gModeling Truncated AR in AD BackgroundMost recently, AR auto-repression was found to be due to the assembly of a FL-AR repressive complex containing lysine-specific demethylase 1 at the intron 2 enhancer [33]. We show here that AR autorepression is not limited to liganded FL-AR, but is effected by TC-AR as well. Unlike the similar ARv567es, co-immunoprecipitation assays revealed no detectable heterodimerization between FL-AR and TC-AR either in a transient transfection assay [2] or in LN/TC-AR (Figure 1E). While this result excludes repression by FL-AR/TC-AR heterodimers, a similar mode of repression by TC-AR homodimers is plausible and warrants further investigation within the context of LN/TC-AR and other truncated AR models. At a minimalistic level, three criteria must be met for a truncated form of AR to be considered a potential surrogate for DHT-bound F.
