FIG. 3.

Content of Grx1, but not Trx1, and total Grx activity are decreased via transfection of Grx1-targeted siRNA into H9c2 cells. (A) Representative Western blot showing Grx1 and Trx1 contents in H9c2 cells transfected with Grx1-targeted siRNA compared to control (scrambled) siRNA and nontransfected cells (none) (see Materials and Methods). (B) Quantification of densitometric analysis (relative density: Grx1/actin) of six Western blots of H9c2 cells transfected with control or Grx1-targeted siRNA (Grx1 content in Grx1 knockdown cells is decreased [i.e., 52 ± 9% (mean ± SEM) decreased compared to control cells (100%)], *p < 0.03. (C) Total Grx activity in H9c2 cells transfected with control or Grx1-targeted siRNA. Cell lysates were added to assay mix containing Na/K buffer, pH 7.5 (0.1 M), GSH (0.5 mM final), NADPH (0.2 mM final), and GR (1 U/mL, final). Cysteine-glutathione mixed disulfide (CSSG, 0.1 mM final) was added to initiate the reaction, and the rate of NADPH oxidation (reflecting the amount of GSSG formed, see Materials and Methods) was measured by monitoring A₃₄₀ for 5 min at 30°C. Grx activity (3.09 ± 0.12 nmol NADPH oxidized/min/mg total protein in control cells; 2.93 ± 0.11 nmol/min/mg in nontransfected cells; and 1.52 ± 0.08 nmol/min/mg in Grx1 knockdown cells) was normalized to the value determined in control cells. Data represent mean ± SEM (n = 4, p < 0.001). Analogous decreases in Grx1 content and total Grx activity were observed when Grx1 was stably knocked down in H9c2 cells using shRNA (see text in Results section).