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Page 6 of 13 Morgan et al. J Cancer Metastasis Treat 2018;4:46 I http://dx.doi.org/10.20517/2394-4722.2018.19

A B C

D E F

G

Figure 2. CAHPV-10 non-invasive prostate cancer cells treated with (A) control (PBS + medium); B: 10 ng/mL HGF; C: 25 ng/mL HGF; D:
50 ng/mL HGF; E: 100 ng/mL HGF for 48 h; F: DU145 cells treated with 10 ng/mL for 48 h as a positive control; G: DU145 cells without
HGF for 48 h as a negative control. Scale bar (shown in A) represents 25µ mol/L for (A-C) and 50µ mol/L for D-G

cant difference in ARF6 levels when CAHPV-10 were compared to the normal prostate cell line PNT2 but
there was a significant difference between non-invasive and invasive cancer cells. ARF6 levels were signifi-
cantly higher in LNCaP (3.9-fold), DU145 (3.3-fold) and PC-3 (4.3-fold) cells compared to CAHPV-10 (P < 0.04,
0.032 and 0.009 respectively). There was no significant difference between the invasive cell lines regardless of
their differing invasive capacities.

To determine whether mRNA levels were translated through to the protein level, we then performed western
blot analysis. Protein bands correlating to ARF6 were evident in all samples [Figure 3A] but densitometry
[Figure 3C] revealed total levels of ARF6 were not significantly different between PNT2 (1.37) and CAH-
PV-10 cells (1.21) yet were significantly increased in the metastatic cell lines LNCaP, 5.92 and PC-3, 3.26 (P <
0.001 and P < 0.034 respectively).

Immunofluorescence shows ARF6 localises to the plasma membrane in aggressive cancer cells
Inactive ARF6 (ARF-GDP) localises to the cytosol and endosomes and when activated (ARF-GTP) translo-
cates to the plasma membrane. Therefore, we carried out immunofluorescence to ascertain the localisation
of ARF6 in all our PCa cells. Figure 4 shows that staining for ARF6 was predominantly localised to the cell