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. 2013 Jun 4;17(6):988–999. doi: 10.1016/j.cmet.2013.04.019

Figure 5.

Figure 5

TRAP1-Induced Downmodulation of SDH Activity Decreases Cell Oxygen Consumption Rate and OXPHOS-Dependent Synthesis of ATP and Prompts Resistance to Stress Stimuli

(A) Representative traces of OCR experiments performed on monolayers of living SAOS-2 cells. Subsequent additions of the ATP synthase inhibitor oligomycin, the uncoupler FCCP, the ETC complex I inhibitor rotenone, and the ETC complex III inhibitor antimycin A were carried out.

(B) ATP levels were measured in mock or shTRAP1 SAOS-2 cells kept in standard culture conditions (bars on the left) or in a focus-forming assay for 15 days (i.e., 1–2 days before cells that did not form foci massively underwent death (bars on the right). Where indicated, cells were treated for 2 hr with the ATP synthase inhibitor oligomycin or the hexokinase inhibitor 5-thioglucose (5TG) in a no-glucose medium to discriminate between ATP produced by OXPHOS and by glycolysis.

(C and D) Representative traces of OCR experiments performed on monolayers of living MEF cells (C) or SAOS-2 cells (D). Experiments were carried out as in (A).

(E) Analysis of the effect of the SDH inhibitor 3-NP on the SQR enzymatic activity of complex II in mitochondria from SAOS-2 cells. 3-NP was added 5 min before starting recordings; 10 mM 3-NP was used to fully inhibit the SDH enzyme.

(F) Cytofluorimetric cell death analysis of SAOS-2 cells starved in a medium without serum for 96 hr with or without the reported concentrations of 3-NP. Viable cells are identified as double negative for propidium iodide and Annexin V-FITC.

(G) Soft agar assay on SAOS-2 cells. Data are reported as fold increase of colony area of mock cells grown with 3-NP compared with mock cells kept without the drug (left) and, separately, as fold increase of colony area of shTRAP1 cells grown with 3-NP compared with shTRAP1 cells kept without the drug (right). In SAOS-2 experiments, mock indicates cells stably transfected with a scrambled shRNA; shTRAP1 indicates cells stably transfected with a TRAP1 shRNA; shTRAP1 + mTRAP1 indicates cells stably transfected with a TRAP1 shRNA and expressing a mouse TRAP1 cDNA. In the experiment with MEFs, cells were stably transfected with either a TRAP1 cDNA or a scrambled shRNA (mock). All bar graphs report mean ± SD values (n ≥ 3); *p < 0.01 with a Student’s t test analysis.