PMC

Histopathology of TRAP1 Tg mice. A–C, prostate tissue samples isolated from WT or TRAP1 Tg mice were examined at 12–15 months of age and histologically scored for hyperplasia (A), atypia of the prostatic epithelium (B), or inflammation (C).

Characterization of TRAP1 transgenic mice. A, total RNA extracted from prostate samples of WT or TRAP1 Tg mice was amplified with primers for TRAP1 by RT-PCR. MW, molecular weight. B, prostate extracts isolated from WT or two independent TRAP1 Tg lines were analyzed by Western blotting (WB). The position of endogenous (Endog) or Tg TRAP1 bands is indicated. Only mouse line 810 expresses Tg TRAP1. C, prostate extracts isolated from WT or TRAP1 Tg mice were immunoprecipitated (IP) with an antibody to Myc and analyzed with an antibody to Myc (top) or TRAP1 (bottom) by Western blotting. Asterisk, not specific. For B and C, molecular mass markers (in kilodaltons) are indicated.

Tg expression of TRAP1 accelerates prostate tumorigenesis. A, prostate tissue sections from Pten^+/−-TRAP1 Tg mice were analyzed histologically by H&E staining and light microscopy. The individual diagnoses are indicated. Specific pathologies are indicated with arrows. DP, dorsal prostate; LP, lateral prostate; AP, anterior prostate; PIN, prostatic intraepithelial neoplasia. B, quantification of prostatic adenocarcinoma (AdCa) in Pten^+/− and Pten^+/−-TRAP1 Tg mice (3–9 months of age). The number of animals examined per condition is indicated. p = 0.04. C–E, Pten^+/−-TRAP1^+/+ or Pten^+/−-TRAP-1^−/− mice were examined histologically for prostate adenocarcinoma (C), prostate hyperplasia (D), or prostatic intraepithelial neoplasia (E). The number of animals examined per condition is indicated.

Histopathology of TRAP1 transgenic mice. A and B, prostate tissue sections from Pten^+/− or Pten^+/−-TRAP1 Tg mice at 3 months of age were analyzed for apoptosis by TUNEL staining (A) with quantification of TUNEL⁺ cells (B). *, p = 0.03. C and D, the experimental conditions are as in A and B, except that prostate tissue sections from Pten^+/− or Pten^+/−-TRAP1 Tg mice at 6 months of age were analyzed for cell proliferation by Ki67 staining (C), and the percentage of Ki67⁺ cells at 3 or 6 months (mo) of age was quantified (D). *, p = 0.02; **, p = 0.005. Data are mean ± S.D. Each point corresponds to an individual measurement from two (B) or four (C and D) independent mice per genotype.

Profiling of TRAP1 Tg mice. A, RNA sequencing. Shown is a heatmap of 30 genes down-regulated (blue) or up-regulated (red) at least 10-fold in prostate samples isolated from WT or TRAP1 Tg mice or, alternatively, Pten^+/− or Pten^+/−-TRAP1 Tg mice, as identified by RNA sequencing. Only genes with expression of any Pten^+/−-TRAP1 Tg sample higher or lower than any sample from other groups are shown. B, RPPA. Shown is a heatmap of 25 proteins up-regulated at least 2-fold in prostate samples of Pten^+/−-TRAP1 Tg compared with Pten^+/− mice. C, prostate samples isolated from Pten^+/− or Pten^+/−-TRAP1 Tg mice were analyzed by Western blotting. Two mice per condition were examined. Molecular mass markers (in kilodaltons) are indicated. D, bioinformatics analysis of predicted prostate cancer networks modulated in Pten^+/−-TRAP1 Tg mice with the most changed genes and proteins highlighted. The impact of transgenic expression of TRAP1 on function is predicted based on Ingenuity Pathway Analysis, activation Z score where available, or overall expression changes of key member genes or directly tested in follow-up experiments.

Phenotype of TRAP1 expression in Pten^+/− prostate epithelial cells. A, primary prostatic epithelial cells were isolated from TRAP1 Tg mice and analyzed for TRAP1 expression by quantitative PCR at the indicated passages (P) in culture. B, Pten^+/− prostate epithelial P8 cells transfected with vector or TRAP1 cDNA were incubated in the presence of staurosporine (1 μm) and analyzed for cell death by direct cell counting. C, Pten^−/− prostate epithelial CapP8 cells or P8 cells transfected as in B were incubated with etoposide (64 nm) and analyzed for caspase-3/7 activity. D—H, P8 cells transfected as in B were analyzed for cell proliferation by direct cell counting on days 5 and 7 after transfection (D), cell invasion across Matrigel-coated inserts (E), ATP/ADP ratio (F), OCR in the presence or absence of the mitochondrial complex I inhibitor rotenone (Rot, 1 μm) (G), or mitochondrial superoxide production (H). *, p = 0.01–0.04; ***, p < 0.0001. H₂O₂ was used as a control oxidative stimulus.