Fig. 5

Denatured Tat-SOD transduced into PC12 cells retained its activity via protein refolding. PC12 cells were treated with or without Tat-SOD (1.5 μM) and incubated in 0, 0.05 or 0.1 mM of CuCl₂ and ZnCl₂, for 2, 4 or 6 h, then cells were lysed. a Enzymatic assay of SOD. SOD-specific activity was analyzed by the pyrogallol method (as described in “Materials and Methods”). b Cellular transduction of SOD by Tat-SOD. Cellular SOD protein expression levels were analyzed by Western blotting. SOD only (without Tat-tagged) was used for the control. c Tat-SOD protected PC 12 cells from superoxide anion attack generated by 70 mM paraquat. After treatment, DNA samples were isolated from PC12 cells and then separated on a 1 % agarose gel. Lane 1, DNA marker; lane 2, cell only; lane 3, paraquat treatment; lane 4, Tat-SOD (1.5 μM) transduced with paraquat. Each experiment was performed in triplicate. All columns displayed as mean ± SEM Significance was calculated compared to controls and p values were calculated by applying Student’s t-test. For the additions of Tat-SOD, statistically very significant differences from added 0.05 or 0.1 mM metals (ZnCl₂/CuCl₂) compared to 0 mM metal control within 2 h (***p < 0.001; ****p < 0.0001, respectively), 4 h (**** p < 0.0001; **p < 0.01, respectively) or 6 h (***p < 0.001; **p < 0.01, respectively); but, statistically no significant difference from 0.5 to 1.0 mM metals added (p > 0.05) within 2, 4 or 6 h. For the no additions of Tat-SOD, statistically no significant differences between added 0.05 or 0.1 mM (ZnCl₂/CuCl₂) metals compared to 0 mM metal control within 2 h (*p < 0.05; **p < 0.01, respectively), 4 h (ns, p > 0.05; **p < 0.01, respectively) or 6 h (***p < 0.001; **p < 0.01, respectively). For the additions added of Tat-SOD within 2, 4 or 6 h with 0 mM metals (ZnCl₂/CuCl₂), statistically significant differences from 2 h compared to 4 or 6 h with no significant differences (p > 0.05), 4 h compared to 6 h with *p < 0.05; with 0.05 mM metals (ZnCl₂/CuCl₂), statistically significant differences from 4 h compared to 2 or 6 h with no significant differences (p > 0.05), 2 h compared to 6 h with *p < 0.05; but, added 0.1 mM metals (ZnCl₂/CuCl₂) within 2, 4 or 6 h statistically no significant differences (p > 0.05)