Figure 5. Knockdown of RHOB affects cell shape and cell migration following overexpression of TC-AR in LN/TC-AR/shR-RHOB.

A LN/TC-AR cells were pre-cultured in hormone-depleted media for 48 hours then treated with Low Dox, High Dox, 1 nM DHT or vehicle for 24 hours. Whole cell lysates were harvested and subjected to western blot analysis. Membranes were probed with α-RHOB or α-actin. B Graphic representation of TC-AR binding sites 100 kb within the region of adjacent to RHOB gene. Arrows above show TC-AR binding sites (+3.8 kb and +47.5 kb downstream of TES). C Androgen-deprived LN/TC-AR/shR-empty and LN/TC-AR/shR-RHOB cells were treated with 1 nM DHT, Low Dox, High Dox or vehicle only. Whole cell lysates were harvested 48-hours post-treatment and subjected to western blot analysis. Membranes were probe with either α-RHOB (top) or α-actin (bottom). D Androgen-deprived LN/TC-AR/shR-RHOB cells were treated with 1 nM DHT, Low Dox, High Dox or vehicle. Images were acquired 48-hours post-treatment. E LN/TC-AR/shR-RHOB cells were pre-cultured in serum free media (SFM) for 24 hours then seeded to migration chambers with various treatments in the presence of SFM for an additional 48 hours after which time fluorescence was detected. Fold induction is relative to untreated control. Prior results for LN/TC-AR (from Figure 3B) are included for comparison. F MTT assay of LN/TC-AR/shR-RHOB in hormone depleted media following treatment with 1 nM DHT, Low Dox, High Dox or vehicle as control showing that DHT independent growth previously shown for LN/TC-AR (Figure 2D) is not inhibited by knockdown of RHOB. Bright field images were acquired with an Olympus microscope using 20× magnification.