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Fig. 2 | Cancer Cell International

Fig. 2

From: Regulation of mitochondrial complex III activity and assembly by TRAP1 in cancer cells

Fig. 2

TRAP1 binding to complex III is metabolically regulated. A, B Fluorescent confocal microscopy analysis of TRAP1 (cy2-donor) and UQCRC2 (cy3-acceptor) (A) and of TRAP1-GFP (donor) and UQCRC2 (cy3-acceptor) (B) in HeLa cells. Dipole–dipole energy transfer from the fluorescent donor to the fluorescent acceptor allowed calculating FRET efficiency (EFRET %) as described in methods section. The overlay images show the InSet area in which FRET has been analyzed. Scale bars: 10 µm. τns values are expressed as mean ± SEM. Two-tailed p values represent the statistical significance based on the Student's t-tests between the τns measured in basal and glucose deprivation conditions (TRAP1 alone versus TRAP1 with UQCRC2). The relative graphs below show the FRET efficiency (percent of control). C, D Representative image of PLA showing the interaction of UQCRC2 with TRAP1 in HeLa cells (C), or in TRAP1-GFP expressing HeLa cells (D), in standard culture conditions and following replacement of standard medium with medium containing galactose in the place of glucose (4.5 g/L) for 4 h. Positive signals of interaction are shown as red dots, nuclei are stained with DAPI (blue). Scale bar: 20 µm. E, F After tetracycline-mediated induction of the expression of TRAP1-directed shRNAs (shTRAP1) or control GFP-directed shRNAs (shGFP) (72 h), or of the expression of the unfused GFP or the TRAP1-GFP fusion protein (24 h), cells were cultured in complete standard medium or low glucose medium (1 g/L) (GD: glucose deprivation) for 4 h and harvested. Intact mitochondria were isolated (see Methods) and lysed for whole complex III isolation. Immunoprecipitates were then subjected to western blot and probed with indicated antibodies. The bar graph in the lower panel shows the ratio between densitometric band intensity of Riske and UQCRC2, expressed as mean ± SEM of two independent experiments. G Representative image of PLA showing the interaction of UQCRC2 with Rieske in shGFP/shTRAP1 HeLa cells, in standard culture conditions and following replacement of medium containing low glucose (1 g/L) (GD: glucose deprivation) for 4 h. Positive signals of interaction are shown as red dots, nuclei are stained with DAPI (blue). Scale bar: 10 µm. The bar graph on the right shows the quantification of the PLA foci as mean spot/cell ± SEM (n = 4). Number above bars represent statistical significance obtained by the Student’s t test. The datapoints referred to the representative images shown on the left are evidenced in red

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