MRC5 cells (1 × 105) were seeded onto coverslips in six-well plates in 10% FBS/EMEM for 24 hours. Cultures were changed to 0.1% FBS/EMEM and treated with either vehicle (4 mM HCl/0.1% BSA) or TGF-β (5 ng/ml) for a total of 6 hours, with MitoTracker (200 nM; Invitrogen) added for the last 30 min. Cultures were then fixed with 3% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 0.1% BSA/10% normal goat serum/PBS, and incubated overnight with HK2 antibody at 4°C. After washing with PBS, HK2 was labeled with 488-Goat-anti-rabbit secondary antibody (Life Technologies) at room temperature for 2 hours, whereas nuclei were stained with 4′,6-diamidino-2-phenylindole at room temperature for 5 min. Fluorescence images were collected on an LSM510 confocal microscope (Carl Zeiss Microimage Inc.). Percentage of colocalization of HK2 with MitoTracker was analyzed by ImageJ software (National Institutes of Health).

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