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. Author manuscript; available in PMC: 2019 May 3.
Published in final edited form as: Mol Cell. 2018 May 3;70(3):502–515.e8. doi: 10.1016/j.molcel.2018.03.029

Figure 6. mTORC1-dependent regulation of SOD1 is important for mitigating oxidative DNA damage in human cells in an ischemic environment.

Figure 6

(A) SOD1 is activated by an ischemic environment. Hep3B cells were subjected to an ischemic condition (1% oxygen and minus glucose) for different times. The activity of SOD1, SOD2 and mTORC1 signaling was followed by in-gel assay and immunoblot. Right panel shows quantification of SOD1 and SOD2 activity, and P-S6K1(T389)(mean ± S.D., N = 3, * p < 0.05, Student’s t-test). HIF1a and p-AMPK were used as ischemic markers.

(B) Ischemic condition activates SOD1 in a T40-dependent manner. Hep3B cells transiently expressing SOD1WT-GFP, SOD1T40A-GFP and SOD1T40E-GFP proteins were subjected to ischemia for 3 hours. SOD1-GFP activity and expression were determined by the in-gel assay and immunoblot, respectively. Bottom, quantification of SOD1 activity (mean ± S.D., N = 3, * p < 0.05, Student’s t-test).

(C) Regulation of SOD1 by mTORC1 is important for cancer cell survival in an ischemic environment. Hep3B cells stably expressing WT and mutant SOD1 under normal or ischemic conditions (−/+ N-acetyl cysteine, NAC) were assayed for cell death by Trypan blue staining after 24 hrs. Data represent mean ± S.D. (N = 3, * p < 0.05, Student’s t-test).

(D) Protective effects of WT and mutant SOD1 against oxidative DNA damage in an ischemic environment. Hep3B cells stably expressing SOD1WT-GFP, SOD1T40A-GFP and SOD1T40E-GFP proteins were subjected to ischemia for 3 hours and analyzed for oxidative DNA damage by immunofluorescence staining with an 8-OxoG antibody. Scale bar, 20 µm.

(E) Quantification of results from Figure 6D. Data represent mean ± S.D. (N = 3, * p < 0.05, Student’s t-test).

(F) Protective effects of WT and mutant SOD1 against DNA damage in an ischemic environment. Hep3B cells stably expressing SOD1WT-GFP, SOD1T40A-GFP and SOD1T40E-GFP proteins were subjected to ischemia for 3 hours and analyzed for DNA damage by immunofluorescence staining with a p-H2A.X(S139)-specific antibody. As a positive control for DNA damage, Hep3B cells were treated with different concentrations of H2O2 for 20 min. Scale bar, 20 µm.

(G) Quantification of results from Figure 6F. Data represent mean ± S.D. (N = 3, * p < 0.05, Student’s t-test).

See also Figures S4 and S5.