Fig. 2. AAV delivered iNOS results in NO production in a dose and time-dependent manner.

(A) Total NO production by TRE-iNOS-transduced differentiated PC12 neurons. NO is produced from TRE-iNOS transduced cells when co-infected with tTA containing vectors dependent upon the infective doses of both TRE-iNOS and the tTA containing vector. NO production increases with time as protein production increases (days 8-14 shown). Data are means (± SEM) from 4 separate wells per group from 2 separate assays. *p < 0.001 compared to cells only at same time point. Nitrite is a stable breakdown product of NO. (B) Regulation of TRE-driven genes by DOX. TRE-eGFP is used as a reporter in 293 cells demonstrating the ability of tTA viruses to drive expression of TRE-delivered genes as well as the inhibitory effect of DOX on TRE-controlled gene expression. A CMV construct is also used as a control promoter for tTA expression (pCMV-tTA-WPRE). No expression is observed in the absence of tTA. Data represent mean fluorescence values (± SEM) using fluorescence and light microscopy images from 3 separate wells of cells per group. *p < 0.001 compared to cells only. (C) NO concentration (nitrite) in 293 cells or primary neurons transduced with TRE-iNOS and MS-tTA showing inhibitory effect of DOX on NO production. Data represent mean NO concentrations (+/- SEM) from 4 separate wells per group. *p < 0.001 compared to cells only, **p < 0.001 compared to addition of MS-tTA, ***p < 0.001 compared to addition of MS-tTA+TRE-iNOS (- DOX) (D) CellTiter-Glo assay showing viability of differentiated PC12 cells transduced with AAVs. Data represent mean percent viability measurements based upon quantitation of ATP (± SEM) from 4 separate wells per group. Data are representative of 3 independent experiments. No significant differences in viability were detected among groups.