Journal: Journal of Biological Chemistry

Article Title: Converging Evidence of Mitochondrial Dysfunction in a Yeast Model of Homocysteine Metabolism Imbalance

doi: 10.1074/jbc.m111.228072

Figure Lengend Snippet: FIGURE 5. Oxidative stress in str4 in the presence of homocysteine and its precursors. A, heat map of up-regulated oxidative stress response genes in the presence of homocysteine and its precursors was generated using Multiexperiment viewer software (MeV version 4.0b). B, flow cytometry measurement of ROS-positive cells. Yeast cells exposed to various conditions for 16 h or hydrogen peroxide for 1 h were harvested and incubated with DHE for 1 h and analyzed by Guava EasyCyte Flow cytometer. Error bars are representative of means S.D. (n 3). C, fluorescence microscopy images were acquired using inverted Nikon epifluorescence microscope in similar experimental conditions. SAM, AdoMet; SAH, AdoHcy; CON, control.

Article Snippet: Cell images were recorded from an inverted epifluorescence microscope (TE200-U, Nikon) under 100 objectives.

Techniques: Generated, Software, Flow Cytometry, Incubation, Fluorescence, Microscopy, Control

Journal: Journal of Biological Chemistry

Article Title: Converging Evidence of Mitochondrial Dysfunction in a Yeast Model of Homocysteine Metabolism Imbalance

doi: 10.1074/jbc.m111.228072

Figure Lengend Snippet: FIGURE 9. Monitoring mitophagy response in str4 strain. A, co-localization of mtGFP and vacuoles. str4 strain containing OM45 protein tagged with GFP as the C-terminal tag was grown in the presence and absence of 5 mM homocysteine and 600 M AdoHcy for 12 h. To stain the vacuole, FM4-64 (at a final concentration of 40 M) was added for 2 h. Live cells were examined under a fluorescence microscope in their respective media to check proper vacuolar staining and Om45-GFP in live cells. Images were acquired with epifluorescence microscope and analyze by ImageJ software. B, detection of free GFP by immune blotting. Cells were collected at 12 h, lysed, and subjected to Western blot analysis with anti-GFP antibody or Nap1p antibody (loading control). The positions of full-length Om45-GFP and free GFP are indicated. C, mitochondrial fission gene expression. Cells were grown in the presence and absence of homocysteine and AdoHcy (SAH) for 16 h. RNA was isolated from these cells and subjected to real time PCR analysis using the gene-specific primers. IPP1 (inorganic pyrophosphatase) gene was used as the internal control (CON) gene for normalization. Error bars are representative of means S.D. (n 2).

Article Snippet: Cell images were recorded from an inverted epifluorescence microscope (TE200-U, Nikon) under 100 objectives.

Techniques: Staining, Concentration Assay, Fluorescence, Microscopy, Software, Western Blot, Control, Gene Expression, Isolation, Real-time Polymerase Chain Reaction