Supplementary Figures

Figure S1. WNT7B is regulated by the AR in CRPC 22RV1 cells. (A) AR occupancy was examined by ChIP-qPCR in the presence or absence of 10 nM DHT for 4h in 22RV1 cells. Site 1 and site 2 are the two AR binding sites identified by ChIP-seq analyses in LNCaP and C4-2B cells (see Fig. 1C). AR occupancy at the PSA enhancer was detected in the absence of DHT (2-fold enrichment over the control), and further enhanced after DHT treatment. (B) RT-qPCR results showing expression of WNT7B remained at a similar level after 10 nM DHT treatment for 16h in 22RV1 cells. (C) AR (full-length and truncated variant) protein levels were examined 3 days after AR siRNA transfection in 22RV1 cells. The AR siRNA (siAR3) was designed to target the AR exon 2, which knocks down both full-length AR and truncated variant AR. (D) WNT7B mRNA levels were examined 3 days after AR siRNA transfection in 22RV1 cells.

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Figure S2. TUNEL assays after WNT7B knockdown. TUNEL assays were performed in LNCaP and C4-2B cells 3 days after WNT7B siRNA transfection in the presence or absence of 10 nM DHT. The fluorographs represent merged nuclear (blue) and TUNEL (green) staining.

Figure S3. Knockdown efficiency of WNT7B shRNA determined by RT-qPCR. C4-2B cells were infected with two different lentiviruses encoding shRNA against WNT7B followed by puromycin selection. Pooled cells were maintained in RPMI 1640 containing 10% FBS and puromycin (1.5 ug/ml). WNT7B expression levels were examined by RT-qPCR before the experiment.

Figure S4. WNT7B is required for 22RV1 cell growth. (A) WNT7B expression in 22RV1 cells was decreased by 50% 3 days after WNT7B siRNA transfection as determined by RT-qPCR. (B) 22RV1 cell proliferation was examined using CCK8 assays 3 days after WNT7B siRNA transfection in the presence or absence of 10 nM DHT. (C) Apoptosis assays with 22RV1 cells showing Caspase 3 and 7 activity 3 days after WNT7B siRNA transfection in the presence or absence of 10 nM DHT. The results are presented as mean ± standard deviations of two independent experiments. **, P < 0.01; ***, P < 0.001, determined by a two-tailed Student’s t-test.

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Figure S5. WNT7B is overexpressed in CRPC tumors. WNT7B expression levels were analyzed in primary androgen-dependent (AD) vs. primary androgen-independent (AI) and localized (Loc) vs. metastatic (Met) prostate tumors using published datasets (NCBI GEO database: GSE2443 and GSE3325). Metastatic tissues were obtained from liver, lymph node, lung, dura and soft tissue metastasis (Varambally et al, Cancer Cell 2005; 8(5):393-406).

Figure S6. β-Catenin distribution in C4-2B cells. Immunostaining of β-catenin (red) in C4-2B cells after overexpression or knockdown of WNT7B in the absence of DHT. DAPI staining (blue) showing nuclei.

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Figure S7. PKCα and PKCδ are direct AR target genes in C4-2B cells. (A) ChIP-seq results showing AR binding events at the PKCα and PKCδ loci. The red arrows indicate the intronic AR-occupied regions. (B) RNA-seq results showing DHT-induced upregulation of PKCα and PKCδ expression in C4-2B cells. The mRNA abundance is recorded in counts per million reads (CPM). Values are mean ± standard deviations of two independent RNA-seq experiments (see Supplementary Table S2).

Figure S8. WNT7B promotes osteoblast differentiation of ST2 and C3H10T1/2 cells. (A) ST2 cells were infected with viruses expressing WNT7B or empty vector and more than 90% infection efficiency was achieved as determined by GFP detection. WNT7B-overexpressing ST2 cells or vector control cells were grown in osteogenic medium. ALP staining was performed on day 7 and von Kossa staining for mineralization was performed on day 21. (B) WNT7B-overexpressing C3H10T1/2 cells and vector control cells were established and examined as described in (A). (C) WNT7B-overexpressing C4-2B cells and empty vector control cells were grown in osteogenic medium. ALP activity and mineralization in C4-2B cells were examined as in (A).

Figure S9. Insoluble WNT7B is bound to the cell membrane. (A) Immunostaining of WNT7B (red) in WNT7B-overexpressing C4-2B cells. DAPI staining (blue) showing nuclei. WNT7B was observed on the cell membrane and in the cytoplasm. (B) The hydrophobic (membrane) protein fraction and hydrophilic (cytoplasmic) proteins were extracted from WNT7B-overexpressing C4-2B cells. WNT7B protein levels of these two fractions were examined by Western blot. (C) ST2 cells were grown in osteogenic medium mixed with conditioned medium from WNT7B-overexpressing cells (LNCaP and C4-2B) or vector control cells (LNCaP and C4-2B) at 1:1 ratio. ALP staining was performed after incubation with conditioned medium for 7 days. Conditioned medium from WNT7B-overexpressing PC cells did not increase ALP activity in ST2 cells.

WNT7B DAPI Merge !"

Figure S10. Mouse ALP, BSP and GAPDH primers are specific for mouse genes. Total RNA was extracted from ST2, C4-2B, and mixed cells respectively. RT-qPCR analyses were performed using primers for mouse ALP, BSP, and GAPDH (mGAPDH). PCR products were run on a 2% agarose gel showing that the primers for mouse ALP, BSP and GAPDH barely amplified the corresponding genes in human C4-2B cells. Specific PCR products were confirmed by Sanger sequencing.

Figure S11. PC-produced WNT7B promotes ST2 osteoblast differentiation. (A) LNCaP cells were stably infected with viruses encoding WNT7B or empty vector (also see Fig. 2E). WNT7B-overexpressing LNCaP cells or vector control cells were co-cultured with ST2 cells in osteogenic medium. ALP staining was performed on day 7 and von Kossa staining for mineralization was performed on day 21. Mouse ALP and BSP mRNA levels were examined by RT-qPCR on day 7. (B) LAPC4 cells were stably infected with viruses encoding WNT7B or empty vector. WNT7B expression levels were examined by Western blot. (C) WNT7B-overexpressing LAPC4 cells or vector control cells were co-cultured with ST2 cells in osteogenic medium. ST2 osteoblast differentiation was examined as in (A).

Figure S12. PC-produced WNT7B promotes C3H10T1/2 osteoblast differentiation. WNT7B-overexpressing LAPC4 cells or vector control cells were co-cultured with C3H10T1/2 cells in osteogenic medium. ALP staining was performed on day 7 and von Kossa staining for mineralization was performed on day 21. Mouse ALP and BSP mRNA levels were examined by RT-qPCR on day 7.