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. Author manuscript; available in PMC: 2014 Jan 10.
Published in final edited form as: Nat Commun. 2013;4:2139. doi: 10.1038/ncomms3139

Figure 3. Mitochondrial Hsp90 regulation of cellular respiration.

Figure 3

(a) PC3 cells were treated with vehicle or Gamitrinib (Gam), solubilized in the indicated increasing concentrations of detergent (NP-40), and insoluble proteins were analyzed by Western blotting with an antibody cocktail to the OXPHOS complex. (b, c) PC3 cells were treated with Gamitrinib (b) or transfected with control non-targeting siRNA (Ctrl) or TRAP-1-directed siRNA (c), and analyzed by Western blotting. None, untreated. (d) PC3 cells were transfected as in (c), treated with the indicated increasing concentrations of H2O2 (μM), and analyzed by Western blotting. (e) PC3 cells were treated with the indicated concentrations of Gamitrinib (Gam, μM) or 17-AAG (10 μM) and analyzed for SDHB activity at the indicated time intervals. NT, not treated. (f) Endogenous Complex II (SDH) was immunoprecipitated from PC3 cells, and analyzed for SDHB activity in the presence of increasing concentrations of recombinant TRAP-1 (μM). Data for panels (e, f) are from representative experiments out of at least two independent determinations. (g) PC3 cells were treated with 17-AAG (5 μM) or the indicated increasing concentrations of Gamitrinib (Gam, μM) and the oxygen consumption rate (OCR) was measured in real time under basal condition and in response to the indicated inhibitors. Arrows indicate the time of drug addition: D, Gamitrinib (Gam) or 17-AAG; O, oligomicyin (1.25 μM); F, FCCP (0.4 μM); A, antimycin (1.8 μM). (h) The OCR was normalized by the number of cells, and the extra-mitochondrial respiration after addition of antimycin was subtracted as background. * p<0.05; ** p<0.01 vs control sample at each state (two-sided unpaired t test). (i) PC3 cells were transfected with control (Ctrl) siRNA or TRAP-1-directed siRNA, treated with Gamitrinib (Gam, μM) or 17-AAG and analyzed for OCR as in (g). (j). Quantification of OCR ratio between: b/o, basal condition (before any addition) and after oligomycin addition; f/o, after FCCP and oligomycin addition; f/b, after FCCP addition and basal condition in PC3 cells transfected with control siRNA (Ctrl) or TRAP-1-directed siRNA. * p<0.05; ** p<0.01 (two-sided unpaired t test). For all OCR experiments, data are representative of two independent experiments carried out in triplicate, mean±sd