Volume 8 issue 11 November 2007
Restoration of Peroxisomal Catalase Import in a Model of Human Cellular Aging
Jay I. Koepke, Kerry-Ann Nakrieko, Christopher S. Wood, Krissy K. Boucher, Laura J. Terlecky, Paul A. Walton, and Stanley R. Terlecky

Supplemental Figure 1: Effect of catalase-SKL expression on endogenous catalase localization. Late passage human (Hs27) diploid fibroblasts were nuclear microinjected with plasmids encoding Xpress™ epitope-tagged catalase-SKL, incubated for 48 h, fixed, simultaneously stained with mouse anti-Xpress™ antibodies followed by CY3-labeled goat anti-mouse secondary antibodies and rabbit anti-catalase antibodies followed by fluorescein isothiocyanate-labeled goat anti-rabbit antibodies, and imaged. The distribution of catalase-SKL appears in panel A and that of total cellular catalase in panel B. A false-color overlay in panel C indicates the extent of co-localization (yellow/orange) of catalase-SKL (red) and total catalase (green) in this cell. Scale bar, 10 µm.

Figure 1 (.jpg)

Supplemental Figure 2: Retroviral expression of catalase in human fibroblasts. A) Xpress™ epitope-tagged catalase-KANL (Cat-KANL) or catalase-SKL (Cat-SKL) was expressed in late passage Hs27 cells using recombinant retroviruses engineered as described in the Materials and Methods. Equivalent numbers of cells were analyzed for the presence of the Xpress™ epitope or glyceraldeyde-3-phosphate dehydrogenase (GAPDH) by western blotting with appropriate antibodies. (-) indicates cells infected with retroviruses expressing a control vector. B) Localization of transduced Xpress™ epitope-tagged catalase-SKL or catalase-KANL in late passage cells was carried out as described in Figure 6 except that rabbit anti-Xpress™ antibodies were followed by rhodamine-conjugated goat anti-rabbit antibodies, and mouse anti-PMP70 antibodies were followed by fluorescein isothiocyanate-conjugated goat anti-mouse antibodies. Scale bar, 10 µm.

Figure 2 (.jpg)

Supplemental Figure 3: Effect of transduced catalase on cellular hydrogen peroxide levels. Late passage human (Hs27) diploid fibroblasts were tranduced with a vector control (A), catalase-SKL (B), or catalase-KANL (C) and the amount of cellular hydrogen peroxide examined using the oxidation-sensitive dye 2´,7´-dichlorofluorescin diacetate. Panel D reflects quantitation (mean fluorescence intensities per unit area ± SEM using National Institutes of Health’s Image J (v1.31) public domain shareware) of the cellular hydrogen peroxide levels seen in the variously treated cells. Scale bar, 10 µm.

Figure 3 (.jpg)