Figure 1. Mitochondrial HSP90 regulation of tumor bioenergetics.

(A) Breast (MCF-7), prostate (PC3, LNCaP), lung (A549, H1473) or brain (glioblastoma, LN229) tumor cell lines were treated with the indicated concentrations of Gamitrinib (Gam) for 5 hr and analyzed for ATP production. Mean±SEM (n=3).

(B) The indicated tumor cell lines were treated with 17-AAG (20 μM) for 5 hr and analyzed for ATP production. Mean±SEM (n=3).

(C) The indicated normal (FF2508, MRC5) or tumor (LN229, PC3, BPH-1) cell lines were incubated with 17-AAG or Gamitrinib (10 μM) for 5 hr and analyzed for ATP production. Mean±SEM (n=3).

(D, E) LN229 cells were treated with the indicated concentrations of Gamitrinib for 5 hr and analyzed for glucose consumption (D) or extracellular lactate content (E). Mean±SEM (n=3); *, p=0.015–0.022.

(F) LN229 cells were plated at the indicated number, treated with vehicle, Gamitrinib or 17-AAG (5 μM) and analyzed for O₂ consumption by a fluorimetric assay. Mean±SEM (n=3), *, p=0.019; **, p=0.001.

(G) PC3 or LN229 cells were incubated with sodium pyruvate (Pyr, 1 mM) in the presence (5, 10 μM) or absence (None) of Gamitrinib for 7 hr and analyzed for ATP production. Mean±SD of replicates (n=2).

(H) LN229 cells were labeled with the fluorescent dye H₂-DCFA (6 μM), treated with Gamitrinib (5–10 μM), and analyzed for changes in fluorescence expression in a luminometer, with or without the antioxidant N-acetyl-L-cysteine (10 mM, NAC). H₂O₂ (5 mM) was used as control. Mean±SEM (n=4).

(I) H₂-DCFA-labeled LN229 cells were treated with 10 μM Gamitrinib for the indicated time intervals and analyzed for changes in ROS production at the indicated time intervals with or without NAC. H₂O₂ was a control. Mean±SEM (n=3).

(J) LN229 transfected with control (Ctrl) or TRAP-1-directed siRNA were analyzed for changes in ATP production or extracellular lactate content (left) or TRAP-1 protein level (right). Mean±SEM (n=3); *, p=0.017; **, p=0.005. See also Figure S1.