Volume 7 issue 1 January 2006
Hypocatalasemic Fibroblasts Accumulate Hydrogen Peroxide and Display Age-Associated Pathologies
Christopher S. Wood, Jay I. Koepke, Hua Teng, Krissy K. Boucher, Sharon Katz, Patrick Chang, Laura J. Terlecky, Irene Papanayotou, Paul A. Walton, and Stanley R. Terlecky

SUPPLEMENTARY MATERIAL:

Supplementary Figure 1: Peroxisomes in normal and hypocatalasemic fibroblasts. Normal human and hypo-catalasemic (Hypocat.) cells were examined for peroxisomal markers by indirect immunofluoresence. The antigen examined in each case is indicated adjacent to the images.

Figure 1 (.tif)

Supplementary Figure 2: DNA damage in human fibroblasts. (A) Oxidative damage to DNA of normal and hypocatalasemic (Hypocat.) fibroblasts was measured using Trevigen Inc.'s FPG FLARE™ assay kit. Briefly, Escherichia coli formamidopyrimidine-DNA glycosidase was used to specifically recognize and excise DNA lesions, which were then unwound and drawn from cells by electrophoresis. Undamaged supercoiled DNA remained in the nucleus of cells. DNA “comet tails” were visualized by staining DNA with the intercalating fluorescent dye, SYBR ^® Green (Molecular Probes). Comet tail length (B) and moment (product of tail length and fraction of DNA in the tail) (C) measurements were made using software suggested by the manufacturer and documented in (Helma C, Uhl J. A public domain image-analysis program for the single-cell
gel-electrophoresis (comet) assay. Mutat Res 2000;466:9-15). Values presented are means +/- SEM for n = 5. Where indicated, (normal) cells were pretreated overnight with 125 μM hydrogen peroxide (+ Hydrogen Peroxide).

Figure 2 (.tif)

Supplementary Figure 3: Hypocatalasemic fibroblasts display a growth stasis but do not stain for the replicative senescence marker, senescence-associated β-galactosidase. In (A), 75,000 normal or hypocatalasemic (Hypocat.) fibroblasts were seeded onto tissue culture plates and their numbers determined at 2, 4, and 6 days afterwards (by counting with a hemacytometer). This experiment was performed twice, and the values presented are means of the cell numbers obtained. In (B), senescence-associated β-galactosidase activity was examined in early and late passage normal fibroblasts as well as in hypocatalasemic (Hypocat.) fibroblasts.

Figure 3 (.jpg)

Supplementary Figure 4: Transduced catalase amasses in organelle pellets of fractionated cells. Anti-catalase western blots of fractionated cell lysates from hypocatalasemic fibroblasts transduced (via Pep-1) with (His-tagged) catalase-SKL. PNS = post-nuclear supernatant (from 1,000 g centrifugation of fractionated cells); S = supernatant (from 10,000 g centrifugation of PNS); P = pellet (from 10,000 g centrifugation of PNS). Note: Only 10% of total PNS was loaded on the gel; the remaining 90% was further fractionated as indicated.

Figure 4 (.tif)

Supplementary Figure 5: An in situ catalase activity gel reveals active (tetrameric) catalase levels in normal and hypocatalasemic (Hypocat.) cells. In (A) the bleached areas indicate metabolized hydrogen peroxide. “+ ATZ” indicates cells pretreated with the catalase inhibitor, aminotriazole. Hypocatalasemic cells transduced (by Pep-1) with catalase-SKL are also indicated. (B) Quantification of the activity gel shown in panel A with a Fujifilm LAS1000plus luminescent image analyzer. Note the units on the ordinate are arbitrary.

Figure 5 (.tif)