Supplementary Materials and Methods

Cell culture

LNCaP cells were acquired from Dr. Leland Chung (Emory University) and PC3 and DU145 cells were purchased from ATCC. LNCaP and PC3 cells were routinely cultured in RPMI 1640 media (Invitrogen) supplemented with 5% FBS at 37°C in a 5% CO2 atmosphere. HOXB13-inducible LNCaP cells (LNCaP-S4) and Tet-on control cells were described previously (1). Briefly, cells were maintained in RPMI media supplemented with 5% tetracycline-free FBS and 600 µg/ml G418 (Invitrogen) and doxycycline (Dox) was used to induce HOXB13 expression. The shHOXB13-LNCaP cells were grown in RPMI media supplemented with 5% FBS and 1µg/ml puromycin as previously described (1). All cultures were fed with fresh medium every 3-4 days.

Transient Transfections

Cells were grown in 5% charcoal dextran-treated (CDT) FBS (Invitrogen) for three days prior to transfections, which were conducted using the Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instruction. pFLAG-HOXB13 has been previously described (2). Deletion mutant of HOXB13 homeodomain (pFLAG-dHD-HOXB13) was constructed by PCR. NF-κB-luc, the NF-κB reporter vector (κB)3-interferon-luciferase, was obtained from Dr. Yongwon Choi (University of Pennsylvania). ZnT4 siRNA was purchased from Santa Cruz Biotechnology, and control siRNA from Bioneer (Daejon, Korea). Six hours after transfection, cells were washed and fed with medium containing 5% CDT-FBS. If needed, cells were treated for 3 hours with TNFα (R&D Systems) at a final concentration of 10ng/ml before cell lysis. Cells were assayed for luciferase activity using the dual luciferase assay system (Promega) and activities were expressed as relative light units (RLU). Transfection experiments were performed in triplicate and results are reported as means ± SDs.

Immunoblotting

Cells were grown to 80% confluence in p60 culture dishes containing 5% FBS-RPMI, and then lysed in protein extraction buffer (1x TBS, 1% NP-40, 0.5% sodium deoxycholate) containing 0.1% SDS and protease inhibitors. Nuclear proteins were extracted using the NE-PER kit (Thermo Scientific), according to the manufacturer’s instruction. Twenty mg of total cell lysates were loaded onto a 10% Bis-Tris gel and proteins were separated using the electroporation system (Biorad). After the proteins were transferred to a PVDF membrane, primary antibodies were applied, followed by incubation with horse peroxidase-conjugated secondary antibodies. Polyclonal antibodies to HOXB13 have been previously described (1). Antibodies to NF-κB/p65, BCL-2, and β-actin were obtained from Santa Cruz Biotechnology. Antibodies to VEGF and MMP-9 were obtained from R&D Systems. Anti- IκBα and IKKα were obtained from Cell Signaling, and antibodies against p21 waf/cip1 from Upstate Biotechnology. Signals were detected and analyzed using a LAS3000 luminescent image analyzer (Fuji, Tokyo).

Immunohistochemistry

Paraffin-embedded human primary PCas were obtained from patients at Chonnam National University Hospital between 1997 and 2005. Specimens from 21 patients comprised 9 ADRPCs and 10 ADPCs. These tumor tissues were derived by radical prostatectomy or transurethral resection of the prostate, as previously described (1). Immunohistochemistry was performed as previously described (1). Briefly, tissues were incubated with antibodies against HOXB13 or IκBα (Cell Signaling) and then with anti-IgG antibodies conjugated to peroxidase (Jackson Laboratories). Colorimetric signals were detected using DAB. Sections were counterstained with hematoxylin for microscopic evaluations. Anti-androgen receptor antibodies (Upstate) were used to confirm tissue integrity. For negative control slides, non-immune rabbit IgG was used. Immunostained slides were evaluated by a pathologist unaware of patient’s clinical detils. Intensities of HOXB13 and IκBα stainings were classified into one of four grades (0, absent; 1, weak; 2, intermediate; and 3, strong). The reliability and reproducibility of this approach has been previously determined by several groups (6-8).

RT-PCR

RNA was extracted from cells as previously described (3). RT-PCR was performed in order to verify expressions of HOXB13, ZnT4, Zip1, and b-actin. The sequences of the primers used were as follows: HOXB13fw, 5’-ccccactgagtttgccttctatc-3’; HOXB13rv, 5’-gcctcttgtccttggtgatgaac-3’; ZnT4fw, 5’-ggagaacttgtaggtggatac-3’; ZnT4rv, 5’-tattagcacaccaacactc-3’; ZnT10fw, 5’-gtcccaaaaggagtcaacat-3’; ZnT10rv, 5’-tttccacttacaagttccc-3’; Zip1fw, 5’-agcttcagagcctccagtgc-3’; Zip1rv, 5’-ccagtacacaggcacgcaag-3’; b-actinfw, 5’-gcaccacaccttctacaatgagc-3’; b-actinrv, 5’-tagcacagcctggatagcaacg-3’. Quantitative RT-PCR was performed as previously described (4). RNA from LNCaP cells was used for standardization purpose. All QRT-PCR experiments were performed twice in triplicate in one 96-well plate. Using the comparative CT method, resulting Ct values were converted to picogram quantities using standard curves. Gene quantities were normalized versus GAPDH and thosereverse transcriptasenon-treated controls were subtracted. These values were then averaged for each triplicate.

References for Supplementary Materials and Methods

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2. Jung C, Kim RS, Zhang HJ, Lee SJ, & Jeng MH (2004) HOXB13 induces growth suppression of prostate cancer cells as a repressor of hormone-activated androgen receptor signaling. Cancer Res 64(24):9185-9192.

3. Jung C, Ou YC, Yeung F, Frierson HF, Jr., & Kao C (2001) Osteocalcin is incompletely spliced in non-osseous tissues. Gene 271(2):143-150.

4. Jung C, et al. (2005) HOXB13 is downregulated in colorectal cancer to confer TCF4-mediated transactivation. Br J Cancer 92(12):2233-2239.

5. Yamashita A, Hayashi N, Sugimura Y, Cunha GR, & Kawamura J (1996) Influence of diethylstilbestrol, Leuprolelin (a luteinizing hormone-releasing hormone analog), Finasteride (a 5 alpha-reductase inhibitor), and castration on the lobar subdivisions of the rat prostate. Prostate 29(1):1-14.

6. Dhanasekaran SM, et al. (2001) Delineation of prognostic biomarkers in prostate cancer. Nature 412(6849):822-826.

7. Rhodes DR, Sanda MG, Otte AP, Chinnaiyan AM, & Rubin MA (2003) Multiplex biomarker approach for determining risk of prostate-specific antigen-defined recurrence of prostate cancer. J Natl Cancer Inst 95(9):661-668.

8. Rubin MA, et al. (2002) alpha-Methylacyl coenzyme A racemase as a tissue biomarker for prostate cancer. Jama 287(13):1662-1670.