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. Author manuscript; available in PMC: 2018 Sep 15.
Published in final edited form as: Cancer Res. 2017 Aug 1;77(18):5054–5067. doi: 10.1158/0008-5472.CAN-17-0106

Figure 6. Mitochondrial superoxide mediates the age-associated decrease in aconitase, complex I and complex V activities.

Figure 6

Following 48 hours treatment of exponentially growing young, adult and old fibroblasts with vehicle control, 0.25 µM GC4419 or 50 MOI of Ad-MnSOD and Ad-Empty, biochemical activity assays for aconitase (6A), Complex I (6B) and Complex V (6C) were performed on whole cell homogenates. Values in (6A, B and C) are expressed as activity (umol/min/mg protein), normalized to the young control group in each panel. Age-associated changes in endogenous SOD activity (Total, MnSOD and CuZnSOD) in exponentially growing young, adult and old fibroblasts (6D) using the SOD assay as described previously (49). (6E) shows changes in SOD activity (CuZnSOD, MnSOD and Total) in exponentially growing dermal fibroblasts from older patients treated with 50 MOI of Ad-MnSOD or Ad-Empty. Values in (6D) and (6E) are expressed as (U/mg protein), normalized to the young control group. (6F) shows changes in Cyanide (CN-) resistant SOD activity in exponentially growing young, adult and old dermal fibroblasts treated with either 50 MOI of Ad-MnSOD or Ad-Empty or 0.25 uM GC4419. SOD activity of GC4419 was retained in full media and in DPBS for 2 weeks at 37°C in a tissue culture incubator (6G) demonstrating the stability of SOD activity. Data plotted in (6G) shows the amount of sample solution required for 50% of maximum inhibition of NBT reduction (49). All experiments are N=3 with three technical and biological replicates per group, **=p<0.05 as compared to the respective young fibroblast group (or as indicated), **** = p<0.001 determined by one-way ANOVA, post-hoc Tukey test using GraphPad prism software.