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. 2016 Jan 21;291(13):6641–6654. doi: 10.1074/jbc.M115.693523

FIGURE 4.

FIGURE 4.

Targeted expression of human catalase to the mitochondria attenuates menadione-induced PRX oxidation in human chondrocytes. Confluent human articular chondrocytes were transduced with an adenovirus expressing mitochondrial catalase (MCAT), a null (empty) vector control, or no virus for 48 h prior to overnight incubation in serum-free medium and addition of 25 μm menadione for the indicated times to induce oxidative stress. Chondrocyte monolayers were incubated in 100 mm NEM for 10 min to alkylate reduced thiols prior to lysis, and cell lysates were prepared in NEM containing lysis buffer prior to immunoblotting. A, immunoblot of chondrocyte mitochondrial and cytosolic fractions showing expression of human catalase protein expression in unstimulated chondrocytes transduced with MCAT adenovirus, a null empty vector, or no virus. B, chondrocyte cell lysates transduced with MCAT adenovirus, a null empty vector, or no virus and then treated with menadione for the indicated times. Hyperoxidized PRXs were identified using the antibody to PRXSO2/3. C, results of densitometric analysis from PRXSO2/3 immunoblots from n = 3 independent donors. D, under nonreducing conditions, immunoblots for total PRX2 allowed for identification of the PRX2 reduced monomer (labeled RM on blots) and the hyperoxidized monomer (labeled HM on blots). PRX2 presented with an oxidized dimer (labeled D on blots). E, under nonreducing conditions, immunoblots for total PRX3 allowed for identification of the PRX3 reduced monomer (labeled RM on blots) and the hyperoxidized monomer (labeled HM on blots). PRX3 presented with an oxidized dimer (labeled D on blots), as well as a lower mixed disulfide band (labeled MD on blots). Solid lines on blots are used to separate different exposures from the same immunoblot. All immunoblots are representative results of n = 3 donors. The data are means ± S.E. normalized to untreated controls. Asterisks represent significant differences compared with control. *, p < 0.05; **, p < 0.01; ****, p < 0.0001 (ANOVA).