Localization of scOxr1 protein in yeast. Fluorescence microscopy was carried out on strain N34 expressing C-terminally GFP-tagged Oxr1 protein. Cells were preloaded with 100 μM MitoTracker Green probe for 30 min prior to collection and mounting in DAPI-containing mounting buffer. Panels: A, DAPI-stained cells; B, MitoTracker; C, Oxr1p-GFP. Immunofluorescence microscopy of strain N-39 expressing Oxr1p-HA was conducted. Panels: D, DAPI; E, anti-HA immunostaining.

Heat and oxidative stress induction of scOXR1. (A) Yeast cells were grown at 22°C to log phase and shifted to 37°C for the times shown (left). Alternatively, cells were grown at 30°C and treated with 0.5 mM H₂O₂ for the times indicated (right). At each time point, cells were harvested and total RNA was analyzed by Northern blotting. Fold induction values are given for each time point relative to the zero-time control. (B) Yeast cells were grown at 22°C to log phase and incubation was continued at 22°C, or cells were shifted to 37°C and incubation was continued for 1 h. Cells were then fixed and stained with anti-HA antibody to visualize Oxr1p-HA. Left side, DAPI staining; right side, anti-HA staining.

Localization of hOXR1 protein in HeLa cells. HeLa cells were preloaded with 100 μM MitoTracker Red probe, fixed, and immunostained with rabbit anti-C7C antibody and anti-rabbit AlexFluor 488 secondary antibody. Panels: A, anti-C7C staining; B, MitoTracker; C, merged images of panels A and B.

(A) H₂O₂-induced accumulation of hOxr1 protein in HeLa cells. Cells were loaded with 100 μM MitoTracker Red for 15 min and then treated with 1 mM H₂O₂ for 15 min or left untreated and allowed to recover in fresh medium for 1 h prior to fixation and staining with anti-C7C antibody. (B) HeLa cells were treated with 1 mM H₂O₂ for 15 min and allowed to recover in fresh medium for the times indicated. Cells were harvested, and crude protein extracts were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-C7C antibody. Samples were also stained with Coomassie as a loading control. (C) Cells were heat stressed at 42°C for 30 min and allowed to recover at 37°C for the times indicated. Western blot assays were conducted as in panel B. Blots were probed with anti-α-tubulin antibody as a loading control. U, unstressed.

Multiple-tissue Northern blot assay of hOxr1. A blot of poly(A)⁺ RNA from several human tissues (Clontech) was probed with ³²P-labeled hOXR1 cDNA.

Mitochondrion-targeted hOxr1 protein complements the peroxide sensitivity of an oxr1Δ::URA3 mutant S. cerevisiae strain. (A) Yeast strains were grown to an optical density at 600 nm of 0.6 and treated with the indicated doses of H₂O₂, and dilutions were plated on synthetic minimal dextrose medium plates without leucine. Colonies were counted after 3 days of growth at 30°C. Strains bearing plasmids expressing either the mitochondrion-targeted (pmt-hOXR1-myc) or the untargeted (phOXR1-myc) form of hOXR1 are compared with wild-type (WT) and vector control (oxr1Δ::URA3) strains. The data shown are representative of three independent experiments. conc., concentration. (B) Protein extracts from oxr1Δ::URA3 strains expressing myc-tagged hOxr1 protein fused to the MTS (mt-hOXR1-myc) or untargeted (hOxr1-myc) were immunoblotted with anti-myc monoclonal antibody 9E10. (C) Yeast cells were loaded with 100 μM MitoTracker Green probe and immunostained for hOxr1-myc proteins with anti-myc 9E10 antibody and AlexaFluor 568 secondary antibody. Cells expressing targeted and untargeted hOxr1 proteins are labeled as in panel B.