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. 2011 Feb 16;6(2):e17218. doi: 10.1371/journal.pone.0017218

Figure 5. Human fibrosarcoma HT1080 cells harbor transcriptionally inactive AR that stimulates cell migration through androgen activation of Rac.

Figure 5

Quiescent fibrosarcoma HT1080 cells were used. Panel A shows the Western blot of cell lysates with the N-20 (upper) or C-19 (lower) anti-AR antibody. Immunoblot analysis of lysates from growing LNCaP or NIH3T3 or 3T3-L1 cells was performed for comparison. In each panel, the levels of tubulin (tub) were determined by Western blot. In B, the cells were transfected with either 3416 or 3424 ARE-luc constructs in the absence or presence of human AR (hAR) expressing plasmid. Cells were made quiescent and then left unstimulated or stimulated for 18 h with 10 nM R1881. Luciferase activity was assayed, normalized using beta-gal as an internal control, and expressed as fold induction. Data from three independent experiments were analyzed. Means and SEM are shown; n represents the number of experiments. The statistical significance of results was also evaluated by Student's t test. In cells co-transfected with hAR and 3416 ARE-luc, the difference in ARE-luc induction between cells challenged with 10 nM R1881 and unstimulated cells was significant (P<0.001). Also significant (P<0.001) was the difference in ARE-luc induction between cells co-transfected with hAR and 3424 ARE-luc and left unstimulated or challenged with 10 nM R1881. Over-expression of hAR in HT1080 cells was verified by Western blot analysis using the C-19 anti-AR antibody (upper inset in B). The levels of tubulin (tub) were also determined (lower inset in B). In C, cells on coverslips were left untreated or treated for 60 min with 10 nM R1881 and then analyzed by IF for AR (left) or nuclei (Hoechst; right). Images are representative of three independent experiments. Scale bar, 5 microM. In D, the cells were wounded, then left unstimulated or stimulated for the indicated time with 10 nM R1881. Casodex (Cx) was used at 1000-fold excess and EHT 1864 at 20 microM. Contrast-phase images are representative of three different experiments.