SUPPLEMENTARY LEGENDS
Supplementary
Figure
S1:
TMPRSS2:ERG
protein
expression
in
PC3c/TMPRSS2:ERG-H cells and VCaP cells. Immunoblotting of protein extracts of
PC3c/TMPRSS2:ERG-H cells and VCaP cells using rabbit monoclonal ERG antibody. HPRT
immunoblotting was used as a loading control.
Supplementary Figure S2: ERG expression in TMPRSS2:ERG-positive and -negative
PCa samples. Relative ERG mRNA expression levels were measured using RT-qPCR in
TMPRSS2:ERG-positive (n=32) and -negative PCa samples (n=20). The results were
normalized to a TMPRSS2:ERG-positive sample. Two independent triplicate experiments
were performed, and results are presented as mean ± SEM.
Supplementary Figure S3:
Ectopic expression of TMPRSS2:ERG in DU145 cells.
Immunoblotting of protein extracts of DU145 cells with either empty pLPCX vectors or
pLPCX-TPRSS2:ERG using rabbit monoclonal ERG antibody. Beta-actin immunoblotting
was used as a loading control.
Supplementary Figure S4: Representative images of gelatin degradation assay. Gelatin
degradation assays were performed as described in Materials and Methods. Degradation areas
are indicated by white arrows.
Supplementary Figure S5: ERG knockdown in H cells. (a) H cells were treated with
pooled control or ERG-specific siRNA, and the ERG knockdown effect was analyzed using
RT-qPCR. The expression level of ERG in H cells treated with control siRNA was normalized
to 1. (b) Western immunoblotting was performed to evaluate the effect of ERG knockdown.
HPRT immunoblotting was used as a loading control.
Supplementary Figure S6: MMP9 and PLXNA2 expression in M cells treated with ERG
siRNA. TMPRSS2:ERG-expressing PC3c M cells were treated with either control siRNA or
ERG siRNA, ERG knockdown was evaluated on the mRNA level using RT-qPCR (a) or on
the protein level using immunoblotting (b). MMP9 (c) and PLXNA2 (d) expression levels
were reduced due to ERG knockdown. The expression levels of ERG, MMP9 and PLXNA2 in
M cells treated with control siRNA were normalized to 1.
Supplementary Figure S7:
MMP9 and PLXNA2 expression in TMPRSS2:ERG-
expressing DU145 cells treated with ERG siRNA. TMPRSS2:ERG-expressing DU145 cells
were treated with either control siRNA or ERG siRNA, ERG knockdown was evaluated on
the mRNA level using RT-qPCR (a) or on the protein level using immunoblotting (b). MMP9
(c) and PLXNA2 (d) expressions were reduced by ERG knockdown. The expression levels of
ERG, MMP9 and PLXNA2 in TMPRSS2:ERG-expressing DU145 cells treated with control
siRNA were normalized to 1.
Supplementary Figure S8: MMP9 knockdown in H cells has no effect on cell migration
or invasion in vitro. H cells treated with pooled MMP9 siRNA showed decreased MMP9
mRNA (a) and secreted MMP9 in the conditioned medium (b). MMP9 knockdown did not
affect H cell migration (c) or invasion (d). Migration and invasion assays were performed as
described in Materials and Methods, as well as in the legend of Figure 7.
Supplementary Figure S9: AR does not regulate PLXNA2 and MMP9 expression in ARexpressing PCa cells. (a) MMP9 and PLXNA2 expression levels in GSE 11428. Wang et al.
treated LNCaP cells with 100 nM DHT for 4 and 16 h. Normalized results are presented as
fold change in expression in DHT-treated cells versus vehicle-treated cells. (b) VCaP cells
were treated with 10nM DHT or vehicle for 16 H, PLXNA2 and MMP9 expression were
evaluated by RT-qPCR. (c) AR binding analysis in PLXNA2 and MMP9 regulatory regions in
LNCaP and VCaP cells.
Supplementary Figure S10: EMT marker expression in TMPRSS2:ERG-expressing
PC3c H, M and L cells compared to control pcDNA cells. CDH1 (E-Cad), VIM (Vimentin),
TWIST1, SNAIL (SNAI1) and SLUG (SNAI2) expression levels were evaluated by RT-qPCR.
Two independent triplicate experiments were performed, and the expression levels of EMT
marks in control pcDNA cells were normalized to 1.
Supplementary Table S1: Primers used in this study.
Supplementary Table S2: 64 common upregulated genes in TMPRS22:ERG-expressing H,
M and L cells.
Supplementary Table S3: 62 common downregulated genes in TMPRS22:ERG-expressing
H, M and L cells.
Supplementary Table S4: Histo-pathological characterization of the primary PCa samples
used in this study.