Figure 2.

Mitochondrial TRAP1 Confers Transforming Potential to Cells

(A and B) Soft agar tumorigenesis assays were performed both in nontransformed cells (i.e., human epithelial prostate RWPE-1 cells) (A) and MEFs (D), stably transfected with either a TRAP1 cDNA or with a scrambled shRNA (mock); and in transformed cells, i.e., human epithelial prostate RWPE-2 cells obtained by v-Ki-Ras expression in RWPE-1 cells (B); cells dubbed shTRAP1a and shTRAP1b were transfected with different TRAP1 shRNAs.

(C) Expression of a mouse TRAP1 cDNA (mTRAP1) insensitive to human-directed shTRAP1 constructs reinstated the capability to form foci in human osteosarcoma SAOS-2 cells stably transfected with TRAP1 shRNAs (shTRAP1).

(D and E) Growth of colonies in soft agar was also assessed in MEF cells (D) or in SAOS-2 shTRAP1 cells (E) stably transfected with a TRAP1 construct lacking the mitochondrial import sequence (ΔNTRAP1). Western immunoblots show TRAP1 expression levels in the different cell types; GAPDH or actin are shown as loading controls. In (D) and (E), the cytosolic localization of ΔNTRAP1 was assessed by subcellular fractionation; caspase-3 and cyclophilin D (CyP-D) are used to verify purity of cytosolic and mitochondrial fractions, respectively. Data are reported as mean ± SD values (n ≥ 3).