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SUPPLEMENTAL FIGURE LEGENDS

Suppl. Fig. 1. Reciprocal gene set enrichment analysis reveals that SPOP F102C, F133V, and F133L mutants have similar effects on the PC transcriptome. We evaluated the reciprocal enrichment of genes up-regulated by transfection in PC cells with each of the three SPOP mutants (F102C, F133L, F133V) against the transcriptomic footprint for each of the remaining SPOP mutants. The results show a strong similarity of the three SPOP mutants (NES ranges 3.31 to 3.64, q=0.05.

Suppl. Fig. 5. The impact of SPOP on AR protein levels in Abl cells is post-translational. Abl PC cells (transfected with control vector or tetracycline-inducible SPOP-wt or the PCassociated SPOP mutants) were treated with vehicle or doxycycline (Dox, 200 ng/ml) for 48 hours. Total RNA was extracted and reverse transcribed. RT-qPCR was used to quantify AR mRNA expression. Relative expression of AR mRNA in the cells was normalized to the expression of β-actin.

Suppl. Fig. 6. Wt-SPOP, but not its PC-associated mutants, suppresses AR protein levels in androgen-dependent PC cells. LNCaP (A) and VCaP (B) cells with tetracycline-inducible

expression of SPOP-wt or PC-associated SPOP mutants were plated in tetracycline-free serum and treated with doxycycline (0, 50, and 500 ng/ml) for 48 hours. Cells were harvested and total cell lysates were prepared. Immunoblot analyses were conducted for the expression levels of AR, HA-SPOP and β-actin in the cell lysates. Densitometric analysis was performed using NIH ImageJ image analysis software.

Suppl. Fig. 7. The interaction between SPOP-wt and AR can occur even in the absence of SRC-3 protein. A. 293T cells, HeLa cells and HeLa SRC-3-/- cells were co-transfected with 1.0 μg of pcDNA3-AR-Flag and 0, 0.8 or 1.5 μg of pcDNA3.1-HA-SPOPwt vector and incubated for 36 hours. At the end of treatment, cells were harvested and cell lysates were prepared. Immunoblot analyses were conducted for the expression levels of AR, SPOP and β-Actin in each sample, as stated in Materials and Methods. B. Immunoblot analysis of wild type SPOP in immunoprecipitates of anti-AR antibody from 293T, HeLa and HeLa SRC-3-/- cells cotransfected with pcDNA3.1-HA-SPOPwt and pcDNA3-AR-Flag for 24 hours. Cells were treated with 250 nM of proteasome inhibitor bortezomib (PS341) for an additional 8 hours prior to harvesting for immunoprecipitation and immunoblot analyses.

Suppl. Fig. 8. Interaction and binding of SPOP-wt to AR is mediated by an SBC motif (a.a.646-a.a.651) located in the Hinge Region of the AR protein. A. Schematic map of AR and its C-terminal truncated variant, ARv7 protein, showing the SBC motif in AR and listing the mutations identified within this motif. B. 293T cells were co-transfected with vectors expressing HA-SPOP-wt and Flag-ARwt or -Flag-AR mutants (-Flag-AR-A646D, -Flag-AR-S648N, -FlagAR-S647F and -Flag-AR-STT648/649/650AAA). The transfected cells were treated with 250

nM of the proteasome inhibitor bortezomib (PS341) for another 8 hours and cells lysates were prepared and utilized for co-IP/immunoblot analysis as in Fig. 2B. Point mutations in the AR SBC motif, specifically A646D, S647F, S648N and STT648/649/650AAA, can abolish the affinity of AR for SPOP-wt. C. 293T cells were co-transfected with vectors expressing HASPOP-wt and Flag-AR-full length (FL) wt or -Flag-ARv7 proteins. The transfected cells were treated with 250 nM of bortezomib (PS341) for another 8 hours and cells lysates were prepared and utilized for co-IP/immunoblot analysis. Contrary to AR-FL, the naturally occurring Cterminally truncated AR variant ARv7, which lacks the SBC motif, did not bind SPOP-wt.

Suppl. Fig. 9. Endogenous SPOP interacts with and regulates the expression levels of the AR protein in PC cells. A. LNCaP, Abl, VCaP, LAPC4, PC3 and DU145 PC cells were treated with 250 nM of bortezomib (PS341) for 8 hours. Cell lysates were incubated with anti-SPOP antibody. The resulting immunoprecipitates were immunoblotted with anti-AR antibody to detect AR protein interaction with endogenous SPOP-wt in LNCaP, Abl, VCaP and LAPC4 PC cells. PC3 and DU145 served as AR-negative controls. B. LNCaP and Abl cells were transfected with non-targeting siRNA (si-NT) and three different SPOP siRNAs (A, B, and C) and incubated for 48 hours. At the end of treatment, cell lysates were prepared and immunoblot analyses were conducted for the expression levels of AR, SPOP and β-Actin in the lysates. Compared to the non-targeting siRNA control (siNT), all three SPOP siRNAs induced significant increase of AR protein levels in these cells. C-D. Expression of AR mRNA transcripts in LNCaP (C) and Abl (D) cells in response to 48 hours of treatment with SPOP siRNAs or si-NT. Total RNA was extracted and AR mRNA levels were determined by RT-qPCR, normalized to 18S RNA levels, and expressed as a % of si-NT AR levels.

Suppl. Fig. 10. The impact of SPOP on AR protein levels in 22Rv1 cells is posttranslational. RTqPCRs for AR-FL (A) and ARv7 (B) mRNA in 22Rv1 cells transfected with si-NT or three different SPOP siRNAs (A, B or C). Neither AR FL nor ARv7 mRNA was increased by silencing SPOP, supporting a post-translational mechanism of action.

Suppl. Fig. 11. Growth promoting effect of PC-associated SPOP mutants in the context of AR axis inhibition (A-B). LNCaP cells with tetracycline-inducible expression of SPOP-wt or the PC-associated SPOP mutants) were plated in medium supplemented with 10% fetal bovine serum (FBS, A) or 10% charcoal-stripped serum (CSS, B), in triplicate wells. In order to maintain SPOP induction, all media were supplemented with doxycycline (50 ng/ml in all wells) and replenished every 48 hrs with fresh medium, serum and antibiotics. Cells were trypsinized and counted on days 4, 8 and 13. LNCaP cells expressing the PC-associated SPOP mutants proliferated faster than cells expressing wt-SPOP. This was observed under regular growth conditions (medium supplemented with 10% FBS, which contains basal androgen levels), as well as under androgen deprivation (medium supplemented with 10% CSS). However, the separation of the growth curves was more pronounced under androgen deprivation conditions, which suggests a more significant contribution of mt-SPOP to cell proliferation under androgen deprivation conditions. C. LNCaP cells with tetracycline-inducible expression of SPOP-wt or the PC-associated SPOP mutants were plated in medium supplemented with 10% FBS in quadruplicate wells and treated with doxycycline (50 ng/ml) without or with 10 µM of enzalutamide (MDV3100) for 96 hours. Cell viability was determined by MTT assay and is presented as % of the control vector. LNCaP

cells carrying the control vector responded to enzalutamide with ~50% inhibition (which is comparable to our prior experience with parental, untransfected LNCaP cells). Cells expressing PC-associated SPOP mutants and treated with DMSO gave a much stronger MTT signal (in agreement with the proliferation-promoting effect of these SPOP mutants), which was somewhat attenuated by enzalutamide, albeit to a smaller respective degree (33-37% inhibition) than in the case of cells carrying the control vector. This finding suggests that mt-SPOP contributes to resistance to AR inhibition, especially as the absolute signal given by enzalutamide-treated SPOP-mutant cells was still higher than the signal of cells carrying the control vector (with or without enzalutamide). In other words, even when treated with enzalutamide, SPOP-mutant cells still proliferate faster than control (endogenous SPOP, or even cells transfected with wt-SPOP) cells that have been treated with or without enzalutamide. Cells transfected with wt-SPOP exhibited suppressed MTT signal even in the presence of DMSO; this was not suppressed any further by enzalutamide treatment, which is expected because of the AR-inhibitory effect of wtSPOP (AR inhibition is already maximal by wt-SPOP and there is no further room for additional AR inhibition by enzalutamide). Thus, this result confirms again that the AR axis is the key transcriptional output of SPOP signaling in PC (if SPOP had major AR-independent effects, then the combination of wt-SPOP with enzalutamide would exert greater inhibitory effect than either alone).

Suppl. Fig. 12. SPOP gene signature scores in human PC specimens correlate strongly with androgen receptor activity (additional patient cohorts). We computed a sum of z-scores for all the genes in the SPOP mutation gene signatures (F133V, F133L, F102C, and combined mutation signature) and for all the androgen-dependent genes (per two androgen-response

datasets: Hieronymus et al (AR activity score 1) and Nelson et al (AR activity score 2)). A. Relative distribution of SPOP gene signature scores and androgen activity scores over primary PC samples from the Taylor et al cohort (GSE21034; R range: 0.39-0.60, all comparisons p