Figure S1. Figure S2. Figure S3.

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Patricia Perry
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1 Figure S1. PSA expression in VCaP was not dependent on the residual androgens in hormonedepleted medium. VCaP or LNCaP cells grown in CSS medium or SFM (serum-free medium) were treated with ethanol (-) or 10 nm DHT (+) for 24h and then immunoblotted for PSA and tubulin (loading control). Figure S2. Abiraterone decreased nuclear expression of AR in VCaP cells. VCaP cells grown in CSS medium were treated with 0 or 10 nm DHT with or without 2 M abiraterone and then analyzed by immunofluorescence with an AR antibody. The photos were taken under 63X objective lens of confocal microscope. DAPI was used to stain nucleus. Figure S3. Progesterone induced PSA expression was blocked by abiraterone. VCaP cells grown in CSS medium were treated with 0, 0.1, or 1 nm progesterone with or without 2 M abiraterone and then immunoblotted.

2 Figure S4. Abiraterone suppressed AR activity in recurrent VCaP xenografts. Tissue samples of recurrent VCaP xenogafts were taken from each mouse pre-treatment and post-treatment with abiraterone (0.5 mg/2d for 8 days) and then subjected to immunohistochemistry using PSA, ERG, AR, or Ki67 antibodies. Representative sections from tumor in one mouse pre- and post treatment are shown. 2

3 Figure S5. Tumor growth was decreased by abiraterone. The Ki67 positive cells pre- and post therapy from tumors in each mouse were counted (average of 10 randomly selected high power fields). Figure S6. Androstenedione induced PSA expression was decreased by indomethacin. VCaP cells in CSS were treated with 0, 1, 10, or 100 nm androstenedione with or without 20 M indomethacin for 24h and then immunoblotted. Figure S7. AR activity was restored in the relapsed VCaP tumors. Sequential tumor samples were taken from the same mouse with a VCaP xenograft right before castration (D), 4 days after castration (C), or at relapse (R) and then subjected to RT-PCR to measure mrna of PSA, TMPRSS2, ERG, or AR. 3

4 Figure S8. AKR1C3 expression was increased in the relapsed VCaP tumors. Sequential tumor samples were taken from the same mouse with a VCaP xenograft right before castration (D), 4 days after castration (C), or at relapse (R) and then subjected to immunohistochemistry using AKR1C3 antibody. Figure S9. Indomethacin did not suppress AR mrna in VCaP cells. VCaP cells grown in CSS medium were treated with 0, 20, or 40 M indomethacin for 24h and then subjected to RT-PCR to measure AR expression. 4

Sequential tumor samples were taken from the same mouse with a VCaP xenograft at

5 Figure S10. AKR1C3 inhibitor indomethacin suppressed AR activity in recurrent VCaP xenografts. Sequential tumor samples were taken from the same mouse with a VCaP xenograft at pre-castration, at relapse, or at relapse after treatment with indomethacin (0.25mg/d) for 2 weeks and then subjected to immunohistochemistry using PSA, ERG, AR, or Ki67 antibody. 5

6 Figure S11. Tumor growth was decreased by indomethacin. The Ki67 positive cells were counted (average of 10 randomly selected high power fields). Significant difference pre- and post-indomethacin (Indo) is indicated. Figure S12. VCaP and VCS2 express higher CYP17A1 protein. Immunoblotting was performed on LNCaP, C4-2, VCaP, or VCS2 cells to measure CYP17A1 expression. CYP17A1 antibody was purchased from Abcam. Figure S13. LNCaP and C4-2 cells express higher CYP11A1 protein. Immunoblotting was performed on LNCaP, C4-2, or VCaP cells to measure CYP11A1 expression. CYP11A1 antibody was purchased from Abcam. 6

7 Figure S14. AR regained nuclear staining in the abiraterone-resistant VCaP xenogarft tumor. Tumor samples were taken from the same mouse with a VCaP CRPC xenograft at preabiraterone, or at abiraterone-resistant stages. Immunohistochemistry was performed using AR antibody. Figure S15. CYP17A1 and CYP11A1 expression in metastatic CRPC. Expression of CYP17A1 and CYP11A1 were compared in relapsed VCaP xenograft and 29 CRPC samples assayed by quantitative real time RT-PCR, with 18S RNA amplified as an internal control. 7

8 Figure S16. The sequence of primers that were used for real-time RT-PCR or PCR. 8

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