Supplementary Materials
List of Supplementary materials:
Supplementary Figures:
Supplementary Figure 1: Strategy for inducible recombination in the prostate
Supplementary Figure 2: Analyses of tumor regression following castration
Supplementary Figure 3: Gene Set Enrichment Analysis (GSEA)
Supplementary Tables:
Supplementary Table 1: List of the primers used in this study
Supplementary Table 2: List of antibodies for used in this study
Supplementary Table 3: Summary of phenotypic analyses
Supplementary Table 4: Differentially expressed genes between Nkx3.1CE2/+; Ptenf/f; BrafCA/+
and Nkx3.1CE2/+; Ptenf/f mouse prostate tumors (p-value<0.01) (included as a separate Excel file)
Supplementary Table 5A: Pathway enrichment analysis for differentially expressed genes
between Nkx3.1CE2/+; Ptenf/f; BrafCA/+ and Nkx3.1CE2/+; Ptenf/f mouse prostate tumors (positive
enrichment) (included as a separate Html file)
Supplementary Table 5B: Pathway enrichment analysis for differentially expressed genes
between Nkx3.1CE2/+; Ptenf/f; BrafCA/+ and Nkx3.1CE2/+; Ptenf/f mouse prostate tumors (Negative
enrichment) (included as a separate Html file)
Supplementary Table 6A: Myc pathway enrichment analysis for differentially expressed genes
between Nkx3.1CE2/+; Ptenf/f; BrafCA/+ and Nkx3.1CE2/+; Ptenf/f mouse prostate tumors. (included as
a separate Html file)
Supplementary Table 6B: Myc pathway enrichment analysis for differentially expressed genes
between RAP+PD treated and Vehicle treated Nkx3.1CE2/+; Ptenf/f; BrafCA/+ mouse prostate
tumors. (included as a separate Html file)
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Supplementary Table 7: Summary of preclinical data
Supplementary Table 8: List of differentially expressed genes between RAP+PD treated and
Vehicle treated Nkx3.1CE2/+; Ptenf/f; BrafCA/+ mouse prostate tumors (p-value<0.01) (included as a
separate Excel file)
2
Supplementary Figure 1
Supplementary Figure S1. Strategy for inducible recombination in the prostate. Relevant mice
have an Nkx3.1CreERT2 allele as well as Ptenflox/flox allele, in which loxp sites flank Exon 5 and/or a
lox-stop-lox activatable oncogenic BrafV600E allele. Recombination is induced by delivery of
tamoxifen at 2-3 months of age. Note that the Nkx3.1CreERT2 allele is null for Nkx3.1, such that
the experimental mice are heterozygous for Nkx3.1.
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Supplementary Figure 2
Supplementary Figure 2. Analyses of response to castration. NPB Mice were induced to form
tumors at 2 months of age and then castrated (or left intact) 1 month later. Shown are whole
mount images of the urogenital regions including prostate (A, B) and H&E images of the tumor
(C, D). The scale bar represents 100 m.
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Supplementary Figure 3
Supplementary Figure S3. Gene Set Enrichment Analysis (GSEA) of genes differentially
regulated between the NPB (Nkx3.1CE2/+; Ptenf/f; BrafCA/+) and NP (Nkx3.1CE2/+; Ptenf/f) mouse
prostate tumors. For pathway analysis, genes comprising the mouse signature genes (i.e., NPB
versus NP) were mapped to their human orthologs and annotated to biological processes. The
positive enrichment scores for the curve of the pathways indicate an enrichment of the overexpressed part of the malignancy signature in the genes that belong to a given pathway.
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Supplementary Table 1: List of the primers used in this study
Purpose and
name of primers
Genotyping
Allele
Nkx3.1CreERT2/+
Ptenflox/lox
Sequences
CAGATGGCGCGGCAACACC
ACTCAAGGCAGGGATGAGC
BrafLSL-V600E/+
TGAGTATTTTTGTGGCAACTG
GCGCGGTCTGGCAGTAAAAAC
GTCATCTTCACTTAGCCATTG
G
CTCTGCTGGGAAAGCGGC
Real time q-PCR
MYC
CCND1
CCND2
CCNB1
GLRX3
EEF1E1
β-ACTIN
Forward
TCCTGTACCTCGTCCGATTC
AGTGCGTGCAGAAGGAGATT
GAGTGGGAACTGGTAGTGTTG
AAGGTGCCTGTGTGTGAACC
CAGTGGTGGAAGTCGGCTC
ACTGAAGCCGGGGAATAAGTA
TCATGAAGTGTGACGTTGACAT
CCGT
Reverse
GGTTTGCCTCTTCTCCACAG
CACAACTTCTCGGCAGTCAA
CGCACAGAGCGATGAAGGT
GTCAGCCCCATCATCTGCG
GCCATGACATCGTTCATCTGT
GCCTGCTTGACTAGATGGGTG
CCTAGAAGCACTTGCGGTGCA
CGATGGAG
Forward
Reverse
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Supplementary Table 2: List of antibodies for used in this study
Antibody
Company
Catalog #
Type
Phospho 4E-BP1(Ser65)
Total 4E-BP1
β -Actin
Androgen Receptor
Phospho Akt (Ser473)
Phospho Akt (Ser473)
Total Akt
Cyclin D1
Cytokeratin 5
Cytokeratin 8
Ki67
Pan Cytokeratin
Phospho eIF4E (Ser209)
Total eIF4E
Phospho p44/42 MAPK
(Erk1/2) (Thr202/Tyr204)
Phospho p44/42 MAPK
(Erk1/2) (Thr202/Tyr204)
Total p44/42 MAPK (Erk1/2)
Phospho GSK-3α/β
(Ser21/9)
Total GSK-3α
Phospho MEK1/2
(Ser217/221)
Total MEK1/2
c-Myc
Phospho p38 MAPK
(Thr180/Tyr182)
RalA
Phospho S6 Ribosomal
Protein (Ser235/236)
Total S6 Ribosomal Protein
Synaptophysin
Cell Signaling
Cell Signaling
Cell Signaling
Santa Cruz
Cell Signaling
Cell Signaling
Cell Signaling
Zymed
Covance
Covance
Novacastra
Dako
Cell Signaling
Cell Signaling
9451
9452
4970
SC-816
3787
4060
9272
13-4500
PRB-160P
MMS-162P
NCL-Ki67p
Z0622
9741
9742
4376
Rabbit
Rabbit
Rabbit
Rabbit
Rabbit
Rabbit
Rabbit
Mouse
Rabbit
Mouse
Rabbit
Rabbit
Rabbit
Rabbit
Cell Signaling
Cell Signaling
9101
Rabbit
Use and dilution
IF
IHC
Western
1:500
1:1000
1:3000
1:500
1:500
1:500
1:1000
1:500
1:500
1:500
1:2000
1:200
1:500
1:1000
1:100
Rabbit
1:500
Cell Signaling
9102
Rabbit
1:1000
Cell Signaling
9331
Rabbit
1:1000
Cell Signaling
9338
Rabbit
1:1000
Cell Signaling
9121
Rabbit
1:500
Cell Signaling
Epitomics
9122
1472-1
Rabbit
Rabbit
1:1000
1:500
Cell Signaling
4511
Rabbit
1:1000
Cell Signaling
3526
Rabbit
1:1000
Cell Signaling
2211
Rabbit
Cell Signaling
Zymed
2217
18-0130
Rabbit
Rabbit
7
1:100
1:1000
1:1000
1:300
Supplementary Table 3: Summary of phenotypic analyses
Genotype(s)
Nkx3.1CreERT2/+;
Pten+/+; Braf+/+
Na
Ageb Phenotypec
Survivald
Weight
(gms)
% KI67
cells
Metastasese
Lymph
node
0/5
Lung
DTCs
0/5
0/5
9
8
Within normal limits
100%
0.19  0.07
2.661  0.16
5
8
Within normal limits
100%
0.28  0.05
P=0.413f
12.08  1.3
P<0.0001
0/5
0/5
0/5
20
9-12
Extensive PIN III and PIN IV, with
some areas of squamous papilloma
100%
0.35  0.07
P=0.1458
22.33  1.22
P<0.0001
0/5
0/10
1/11
21
2-7
Microinvasive adenocarcinoma with
areas of adenomatous, squamous,
and solid spindle cell components;
other areas of complex papillar tumor
with adenosquamous and goblet cell
components
1.8  0.3
P<0.0001
36.25  3.36
P<0.0001
3/5
3/10
2/10
(N)
Nkx3.1CreERT2/+;
Pten+/+;
BrafCA/+
(NB)
Nkx3.1CreERT2/+;
Ptenflox/flox;
Braf+/+
(NP)
Nkx3.1CreERT2/+;
Ptenflox/flox;
BrafCA/+
(NPB)
4 mths 
0.8
P<0.0001
NOTES:
a) N is the total number of mice analyzed
b) Age refers to the time of analyses after induction with Tamoxifen (or treatment with corn oil); mice were induced at 2 months of age.
c) Phenotype refers to the pathological description of this histological phenotype according to the classification of Park et al. 2002.
d) Survival refers either to the % of mice alive at the time of analyses or to the average age of lethality represented in months.
e) Tissue metastases were scored by analyses of H&E and with two IHC markers (AR and Pan-Cytokeratin); Disseminated tumor cells
(DTCs) were evaluated from bone using a PCR based assay.
f) All P values compare the experimental group (as indicated) to the control group.
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Supplementary Table 7: Preclinical treatment data
Proliferation
Genotype
Treatment N Weight
(grams)
(%)
CreERT2/+;
Nkx3.1
Ptenflox/flox;
BrafCA/+
(NPB)
Vehicle
10
RAP
6
PD
6
RAP
+ PD
10
0.43
 0.18
0.60
 0.08
P = 0.50
0.74
 0.08
P = 0.22
0.09
 0.02
P<0.0001
9
Lung
Mets
36 3.3
3/10
Disseminated
Tumor Cells
2/10
23  2.7
P = 0.02
ND
ND
13  1.03
P = 0.0003
ND
ND
10
1.12
P<0.0001
1/10
0/10