Figure 5.

Transcriptional control of metabolism by mTOR in AR-null PCa cells. (A) Status of the mTOR signaling pathway activity following treatment with vehicle (V), rapamycin (R), or torin 1 (T) in PC3 and DU145 cells in the absence of androgens. Tubulin is shown as a loading control. (B) Detection of nuclear mTOR by Western blotting in LNCaP, PC3, and DU145 cells. Lamin B1 and tubulin are shown as loading controls for nuclear and cytoplasmic extracts, respectively. (C) ChIP-qPCR assessment of mTOR recruitment to DNA regulatory regions of metabolic genes in PC3 and DU145 cells. Results are shown as the average of three independent experiments. (D) qRT–PCR assessment of metabolic gene expression related to glycolysis (left) or mitochondrial and lipid metabolism (right) following 48 h of treatment with vehicle, rapamycin (rapa), or torin 1. (E) Glucose consumption and lactate production measured from the media of PC3 and DU145 cultured cells following a 48-h treatment with rapamycin, torin 1, or vehicle (control). (F) Cell number determination of PC3 and DU145 cells following a 48-h treatment with rapamycin, torin 1, or vehicle (control). (G) Migration assay for PC3 and DU145 cells with or without treatment with the mTOR inhibitor torin 1. Values in D–G represent mean ± SEM of at least three independent experiments performed in triplicate. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.