Mitochondria are the major determinant of intracellular disulfide formation. (a) Fluorescent imaging of protein disulfides in Chang liver cells after treatment with CCCP for 4 h. (b) Effect of different mitochondrial inhibitors on disulfide-containing proteins (filled bars) and superoxide generation (open bars) measured by dihydroethidium fluorescence in bovine aortic endothelial cells (BAECs): a, control; b, rotenone; c, myxothiazol; d, TTFA; e, ATM; and f, CCCP. Protein disulfide staining was semiquantified with fluorescence microscopy and IMAGEJ software. (c) Effect of inhibitors of catalase [3-amino-1,2,4-triazole (3-AT)], GPx [β-mercaptosuccinic acid (MS)], and a GPx-mimetic, ebselen, on protein disulfide content. (d) Effect of antioxidant enzyme overexpression on protein disulfide formation. Cells were infected with a 25 multiplicity of infection (MOI) adenovirus containing the following cDNAs: MnSOD, manganese superoxide dismutase; CAT, wild-type catalase; and MitoCAT, catalase targeted to mitochondria. (e) Protein disulfide formation decreases in Rho₀ cells devoid of functional mitochondria (26). (f) Biotin-labeled disulfide-containing proteins. Protein disulfides were labeled with either MTSEA-biotin or biotinylated iodoacetamide (BIAM) and then detected with streptavidin-conjugated HRP. Lane 1, control; lane 2, CCCP; lane 3, ATM. Cells were treated with mitochondrial inhibitors for 8 h.

Effect of mitochondrial inhibitors on various growth factor receptors. Cells were treated with mitochondrial inhibitors for 8 h before assays. (a) Uptake of Alexa 488-labeled EGF by Chang liver cells or AcLDL by HPAECs. Nuclei were counterstained with DAPI. (b) Time-dependent decrease of AcLDL uptake (filled circles) and mitochondrial membrane potential (open circles) in HPAECs after treatment with CCCP. Mitochondrial potential was measured by fluorescence of JC-1 dye expressed as the ratio of emission at 590 nm and at 536 nm. (c–e) To measure cell-surface receptor autophosphorylation, cells were incubated with 100 ng/ml EGF (c), 25 ng/ml IGF-1 (d), or 25 ng/ml bFGF (e) for 5 min before lysis and analysis by immunoblot. (f) Localization of EGFR, IGF-1Rβ, and PDI to the cell lysate or cell surface and effect of mitochondrial inhibitors.

Effect of mitochondrial inhibitors on specific protein disulfides. (a) Immunoblot of disulfide-containing and total actin and CD98. (b) Heterodimer formation of CD98 in mitochondrial inhibitor-treated cells. (c) Immunoblot of multimeric VWF in HPAECs after mitochondrial inhibitor treatment. Lane 1, control; lane 2, CCCP; lane 3, ATM; lane 4, TTFA; lane 5, dehydroepiandrosterone (DHEA); lane 6, buthionine sulfoximine (BSO). (d) Immunoblot of endoglin on a nonreducing gel. Antibody used was a mouse monoclonal antibody (P4A4) or a rabbit polyclonal antibody (H300). (e) Immunofluorescence (green) of endoglin, PECAM, and VWF in HPAECs after treatment with mitochondrial inhibitors. The Golgi was stained with Alexa 350-labeled wheat germ agglutinin (WGA) (blue) in the endoglin experiment, whereas nuclei were stained with DAPI (blue) in the PECAM and VWF experiments. (f) GRP78 and GRP94 protein induction by mitochondrial inhibitors, tunicamycin (TUNI) or thapsigargin (THAS).