Ent regression to estimate the mean slope of regression lines for the relationship of change in HR to change in MAP within animals in a treatment group, and compared mean slopes among the treatments. Significance was accepted at P values 0.05. ResultsDownregulation of nNOS in HEK cells with AAVp-nNOSshRNAWe first sought to establish that an shRNA that we developed as described above efficiently downregulated2012 The Authors. The Journal of PhysiologyC2012 The Physiological SocietyL.-H. Lin and othersJ Physiol 590.expression of nNOS in vitro. In human embryonic kidney cells (HEK293 cell line), which do not express nNOS, we induced expression of the protein by exposing the cells for 48 h to AAVp-nNOScDNA. Utilizing real time RT-PCR we found that treated HEK cells demonstrated nNOS mRNA expression after this treatment (Fig. 1A). We saw 94 Cyanein custom synthesis reduction of induced nNOS mRNA when the cells were also incubated with AAVp-nNOSshRNA. Consistent with RT-PCR results, Western blot analysis also showed induced expression of nNOS protein after HEK cells were incubated with AAVp-NOScDNA and reduction of nNOS after exposure of the cells to AAVp-nNOSshRNA (Fig. 1B).Downregulation of nNOS in NTS with AAV2nNOSshRNATo test efficacy of transduction in vivo, we first introduced the AAV2nNOSshRNA unilaterally (n = 6) into the NTS and TAPI-2MedChemExpress TAPI-2 documented marked loss of nNOS immunofluorescence at the site of injection 2 weeks later (Fig. 2), when compared with that in animals (n = 5) in which PBS had been injected into the NTS (Table 1). We also observed a significant decrease (P < 0.00001) in nNOS-immunoreactivity (IR) in the contralateral side of the NTS with grey value of 12.5 ?1.6. The decrease in nNOS-IR in the contralateral NTS most likely was due to transport of AAV2nNOSshRNA from the injection side to the contralateral side as AAV2 vectors undergo anterograde and retrograde transport (Lin et al. 2011). Further, we observed a significant decrease (P < 0.00001) in the number of cells that were positive for nNOS-IR in the ipsilateral NTS (Table 2). The number of cells that were positive for nNOS-IR in the contralateral NTS also was significantly decreased (P < 0.00001), to 3.6 ?1.1 per section of NTS. Similar to the results in our previousFigure 1. Expression of nNOS was reduced in HEK293 cells by AAVp-nNOSshRNA A, real time RT-PCR results show that AAVp-nNOSshRNA led to 94 reduction (mean of 3 set of experiments) in induced nNOS mRNA (column AAV2shRNA). A control short hairpin RNA did not affect nNOS expression (data not shown). B, Western blot shows that a decrease in nNOS protein was also observed by AAVp-nNOSshRNA (lane AAV2shRNA). The nNOS protein band (160 kDa) is indicated by the arrow on the left side of the blot.study (Lin et al. 2011), injection of PBS did not change nNOS-IR in the NTS, while injection of AAV2nNOScDNA increased nNOS-IR in the NTS, when compared with that of un-injected rats. As mentioned above, we have shown that AAV2 vectors undergo anterograde and retrograde transport (Lin et al. 2011). Therefore, we also examined the NG, RVLM, NA, and CVLM to determine if AAV2nNOSshRNA changes nNOS expression in these areas. Compared to that of PBS injected controls, we saw a significant decrease (P < 0.00001) in the number of nNOS-IR positive cells in the ipsilateral NG (Fig. 2 and Table 2), when compared to that of PBS injected group. The number of nNOS-IR positive cells in the contralateral NG also showed a significant decrease (P < 0.00001) to 21 ?5.Ent regression to estimate the mean slope of regression lines for the relationship of change in HR to change in MAP within animals in a treatment group, and compared mean slopes among the treatments. Significance was accepted at P values 0.05. ResultsDownregulation of nNOS in HEK cells with AAVp-nNOSshRNAWe first sought to establish that an shRNA that we developed as described above efficiently downregulated2012 The Authors. The Journal of PhysiologyC2012 The Physiological SocietyL.-H. Lin and othersJ Physiol 590.expression of nNOS in vitro. In human embryonic kidney cells (HEK293 cell line), which do not express nNOS, we induced expression of the protein by exposing the cells for 48 h to AAVp-nNOScDNA. Utilizing real time RT-PCR we found that treated HEK cells demonstrated nNOS mRNA expression after this treatment (Fig. 1A). We saw 94 reduction of induced nNOS mRNA when the cells were also incubated with AAVp-nNOSshRNA. Consistent with RT-PCR results, Western blot analysis also showed induced expression of nNOS protein after HEK cells were incubated with AAVp-NOScDNA and reduction of nNOS after exposure of the cells to AAVp-nNOSshRNA (Fig. 1B).Downregulation of nNOS in NTS with AAV2nNOSshRNATo test efficacy of transduction in vivo, we first introduced the AAV2nNOSshRNA unilaterally (n = 6) into the NTS and documented marked loss of nNOS immunofluorescence at the site of injection 2 weeks later (Fig. 2), when compared with that in animals (n = 5) in which PBS had been injected into the NTS (Table 1). We also observed a significant decrease (P < 0.00001) in nNOS-immunoreactivity (IR) in the contralateral side of the NTS with grey value of 12.5 ?1.6. The decrease in nNOS-IR in the contralateral NTS most likely was due to transport of AAV2nNOSshRNA from the injection side to the contralateral side as AAV2 vectors undergo anterograde and retrograde transport (Lin et al. 2011). Further, we observed a significant decrease (P < 0.00001) in the number of cells that were positive for nNOS-IR in the ipsilateral NTS (Table 2). The number of cells that were positive for nNOS-IR in the contralateral NTS also was significantly decreased (P < 0.00001), to 3.6 ?1.1 per section of NTS. Similar to the results in our previousFigure 1. Expression of nNOS was reduced in HEK293 cells by AAVp-nNOSshRNA A, real time RT-PCR results show that AAVp-nNOSshRNA led to 94 reduction (mean of 3 set of experiments) in induced nNOS mRNA (column AAV2shRNA). A control short hairpin RNA did not affect nNOS expression (data not shown). B, Western blot shows that a decrease in nNOS protein was also observed by AAVp-nNOSshRNA (lane AAV2shRNA). The nNOS protein band (160 kDa) is indicated by the arrow on the left side of the blot.study (Lin et al. 2011), injection of PBS did not change nNOS-IR in the NTS, while injection of AAV2nNOScDNA increased nNOS-IR in the NTS, when compared with that of un-injected rats. As mentioned above, we have shown that AAV2 vectors undergo anterograde and retrograde transport (Lin et al. 2011). Therefore, we also examined the NG, RVLM, NA, and CVLM to determine if AAV2nNOSshRNA changes nNOS expression in these areas. Compared to that of PBS injected controls, we saw a significant decrease (P < 0.00001) in the number of nNOS-IR positive cells in the ipsilateral NG (Fig. 2 and Table 2), when compared to that of PBS injected group. The number of nNOS-IR positive cells in the contralateral NG also showed a significant decrease (P < 0.00001) to 21 ?5.