(A–F) Human prostate TWIST1/2 expression increases with age. Representative IHC images of TWIST1/2 expression (brown) in normal human prostate tissue specimens derived from patients aged (A and B) 30, (C and D) 45, and (E and F) 80 years. Magnification is ×10 in A, C, and E, and ×20 in insets in B, D, and F. Scale bars: 100 μm. (G) Graph depicts percentage of samples positive for TWIST1/2 in normal human prostate tissue. IHC scores of ≥70 were considered positive. Each dot represents an individual patient with positive TWIST1/2. **P < 0.01 by χ² test, as shown in Supplemental Figure 3A. (H–J) Endocrine GH induces prostate cell EMT and proliferation. Western blot of EMT markers in (H) HPrEC line 1, (I) HPrEC line 2, and (J) PNT2. Cells were treated with indicated GH doses and analyzed 24 hours later. Representative blots from at least 2–4 independent experiments are shown. ImageJ quantification of Western blots is depicted in Supplemental Figure 3, C–E. For H, the Western blot membrane from Figure 2D was stripped and re-probed for TWIST2 and Ki67; the GAPDH loading control is the same. (K–M) Migration of (K) HPrEC line 1, (L) HPrEC line 2, and (M) PNT2 cells treated with GH. Cells were plated into migration chambers with GH (100 ng/mL) or left untreated (control); 24 hours later, cells were stained with crystal violet, imaged (magnification, ×100), and the number of migrated cells was assessed. Results shown are mean ± SEM. Each dot represents results of 3 separate experiments performed in duplicate. Results are shown as percentage of control, but statistical testing was performed on raw numbers. Results in K–M were analyzed by 2-tailed Student’s t test. **P < 0.01 versus control.