Figure 2. Effect of SOD1 or SOD2 knockdown on the death of HT22 cells induced by glutamate or hydrogen peroxide.

A. HT22 cells were stably transfected with the SOD1 or SOD2 shRNA plasmid or with a scrambled non-targeting shRNA plasmid as described in Materials and Methods. Cell extracts were prepared and subjected to Western blotting of SOD1 and SOD2. Membranes were stripped and re-probed for GAPDH as a loading control. Shown are results from a representative experiment. B. Protein level was quantified using the Scion image software (Scion Corporation, Frederick, MD) and normalized as the ratio to GAPDH. C. Mitochondrial and cytosolic fractions were isolated from control cells as well as SOD1- and SOD2-knock down cells. The SOD enzymatic activity in each fraction was measured and normalized as a ratio relative to the control activity. D and E. Control and SOD-knockdown cells were treated with glutamate (D) or hydrogen peroxide (E), respectively, at indicated concentrations for 24 h. F. HT22 cells were transfected with the SOD1 siRNA or with a scrambled non-targeting siRNA as described in Materials and Methods. Cell extracts were prepared and subjected to Western blotting of SOD1 and SOD2. Membranes were stripped and re-probed for GAPDH as a loading control. Protein levels were quantified and normalized as the ratio to GAPDH. G and H. HT22 cells were transfected with scrambled siRNA or SOD1 siRNA. Forty-eight h later, cells were treated with glutamate (G) or hydrogen peroxide (H), respectively, at indicated concentrations for 24 h. Cell viability was determined using the MTT assay. Vertical error bars indicate standard deviation (S.D.), with N = 5 for each treatment group. The experiment was repeated over three times, and similar observations were made (a representative data set is shown). * P < 0.01, **P < 0.001.