Fig 1.
Growth rates of VCaP-T and VCaP-CT during and after establishment.
(A) Growth rates of increasing passages (p.) of VCaP-T and VCaP-CT cells during long-term cell culture. (B) Growth rates of VCaP-T (p.84.) and VCaP-CT (p.73.) cells in hormone-depletion (control) and increasing nanomolar (nM) concentrations of T. The cell numbers were measured after 7 days growth and compared relative to the cell number at day 0 (dotted line). Data are represented as mean ± SD and three separate replicates were used.
Fig 2.
Measurement of androgen receptor (AR) protein and prostate specific antigen (PSA) levels.
VCaP-T and VCaP-CT cells were treated with the indicated concentrations of testosterone (T), 10 μM bicalutamide (bic), 10 μm enzalutamide (enz) for 72 h, or were hormone-depleted for 72 h (control). (A) Analysis of AR protein levels by Western blotting. Beta-actin was used as a loading control. Control of VCaP-T (top-left corner) was additionally shown in the same Western blot as VCaP-CT to address the difference. Different rows represent different blots. Whole blots with molecular mass markers are presented in S1 Fig. (B) Analysis of the amount of secreted PSA levels. Data are represented as mean ± SD and three separate replicates were used.
Fig 3.
The effect of antiandrogens bicalutamide or enzalutamide on the growth of VCaP-T and VCaP-CT cells.
(A) Growth of VCaP-T in the presence of 0.1 nM T and increasing micromolar (μM) concentrations of antiandrogens. (B) Growth of VCaP-CT in the presence of 0.1 nM T and increasing concentrations of antiandrogens. (C) Growth of VCaP-T in the presence of 10 nM T and increasing concentrations of antiandrogens. (D) Growth of VCaP-CT in the presence of 10 nM T and increasing concentrations of antiandrogens. The cell numbers were measured after 7 days treatments and compared relative to the control (no antiandrogen) sample. Data are represented as mean ± SD and three separate replicates were used.
Fig 4.
Schematic illustration of the categorization of differentially expressed genes in VCaP-T and VCaP-CT cells.
(A) AR-associated genes were identified by treating VCaP-T cells with low T concentration (0.1 nM) for 48 h. These AR-associated genes were further categorized to genes that were adaptive (expression restored) or non-adaptive (expression non-restored) in VCaP-CT cells. Whether the AR-association of the genes was lost or maintained during the acquisition to low T concentration, was assessed by treating VCaP-CT cells with 10 nM T for 48 h. (B) Genes that were not T-regulated in VCaP-T cells were categorized to non-AR-associated genes. Whether the AR-association of the genes was gained or not during the acquisition to low T concentration, was assessed by treating VCaP-CT cells with 10 nM T for 48 h.
Fig 5.
Heatmap illustration of expression levels of 418 AR-associated genes in VCaP-T and VCaP-CT cell lines upon testosterone level changes. Expression levels are scaled and centered by row. Using hierarchal clustering with Euclidean distances genes were clustered into 9 clusters with different characteristics. Genes with association to progression-free survival are pointed.
Fig 6.
Non-AR-associated genes gaining AR-association in VCaP-CT.
Heatmap of 151 non-AR-associated genes that gained AR-association in VCaP-CT and their expression levels in VCaP-T and VCaP-CT cell lines upon testosterone level changes. Expression levels are scaled and centered by row. Using hierarchal clustering with Euclidean distances genes were clustered into 4 clusters with different characteristics. Genes with association to progression-free survival are pointed.
Table 1.
Association between expression levels of androgen responsive genes and prostate cancer progression-free survival.