Cells were induced with the indicated reagents at 24 h after transfection

the control LCLs. When the two AD subgroups were compared, AD-A LCLs were found to have a significantly higher basal ECAR than the AD-N LCLs, and the significant decrease in ECAR with increasing DMNQ concentrations was significantly greater in magnitude for AD-A LCLs as compared to AD-N LCLs . These data demonstrate that, in general, AD LCLs are more dependent on glycolysis for energy production with this dependency being particularly significant for the AD-A LCLs as compared to the AD-N LCLs. Overall, glycolytic reserve capacity was found to be higher in the AD LCLs as compared to the control LCLs . Glycolytic reserve capacity was found to change significantly as DMNQ increased peaking at 5 mM DMNQ and then decreasing at higher DMNQ concentrations. There was a significant DMNQ by group interaction due to the fact that glycolytic reserve capacity was greater for the AD LCLs as compared to the control LCLs at lower DMNQ concentrations but decreased to become more alike as DMNQ concentration increased. When we examined the two AD subgroups separately, we found that the glycolytic reserve 6099352 capacity for the AD-N LCLs was not significantly different than the control LCLs, and the significant change in glycolytic reserve capacity with increasing DMNQ was not 19094061 different between groups. However, the AD-A LCLs did demonstrate a significantly higher glycolytic reserve capacity as compared to the control LCLs . Glycolytic reserve capacity changed significantly as DMNQ increased with this change significantly different across the two LCL groups. Glycolytic reserve capacity was greater for the AD-A LCLs as compared to the control LCLs at lower DMNQ concentrations but decreased to become more alike as DMNQ concentration increased. Comparing the two LCL groups revealed that the AD-A LCLs exhibited a significantly higher glycolytic reserve capacity as compared to the AD-N LCLs . Glycolytic reserve capacity changed significantly as DMNQ increased although this pattern of change was not significantly different across the two groups. Inhibition of UCP2 Affects AD-N and AD-A LCLs Differently In order to determine the differential ability of the AD LCL subgroups to adapt to intramitochondrial oxidative stress at the inner mitochondrial membrane, we used genipin to inhibit UCP2, the key protein on the inner mitochondrial membrane which regulates proton leak to reduce ETC generated oxidative stress. 8 Mitochondrial Dysfunction in Autism Cell Lines For this set of experiments, we used only two concentrations of DMNQ, 0 mM and 10 mM. Overall, LCLs order Aglafoline exposed to genipin exhibited higher ATP-linked respiration than unexposed LCLs . ATP-linked respiration was also overall higher for the AD-A than the AD-N LCLs. Interestingly, there was a DMNQ by genipin interaction such that ATP-linked respiration did not increase with DMNQ for the LCLs unexposed to genipin, but it increased significantly with DMNQ in the LCLs exposed to genipin. Overall, proton leak respiration was greater for the LCLs exposed to genipin as compared to the unexposed LCLs . It was also higher in the AD-A LCLs compared to the AD-N LCLs and in the LCLs exposed to DMNQ compared to the LCLs not treated with DMNQ. There was a DMNQ by genipin interaction because the increase in proton leak respiration associated with DMNQ was greater for the LCLs exposed to genipin as compared to those unexposed to genipin. Genipin also resulted in a more significant increase in proton leak respiration for the AD-N LCLs as the control LCLs. When the two AD subgroups were compared, AD-A LCLs were found to have a significantly higher basal ECAR than the AD-N LCLs, and the significant decrease in ECAR with increasing DMNQ concentrations was significantly greater in magnitude for AD-A LCLs as compared to AD-N LCLs . These data demonstrate that, in general, AD LCLs are more dependent on glycolysis for energy production with this dependency being particularly significant for the AD-A LCLs as compared to the AD-N LCLs. Overall, glycolytic reserve capacity was found to be higher in the AD LCLs as compared to the control LCLs . Glycolytic reserve capacity was found to change significantly as DMNQ increased peaking at 5 mM DMNQ and then decreasing at higher DMNQ concentrations. There was a significant DMNQ by group interaction due to the fact that glycolytic reserve capacity was greater for the AD LCLs as compared to the control LCLs at lower DMNQ concentrations but decreased to become more alike as DMNQ concentration increased. When we examined the two AD subgroups separately, we found that the glycolytic reserve capacity for the AD-N LCLs was not significantly different than the control LCLs, and the significant change in glycolytic reserve capacity with increasing DMNQ was not different between groups. However, the AD-A LCLs did demonstrate a significantly higher glycolytic reserve capacity as compared to the control LCLs . Glycolytic reserve capacity changed significantly as DMNQ increased with this change significantly different across the two LCL groups. Glycolytic reserve capacity was greater for the AD-A LCLs as compared to the control LCLs at lower DMNQ concentrations but decreased to become more alike as DMNQ concentration increased. Comparing the two LCL groups revealed that the AD-A LCLs exhibited a significantly higher glycolytic reserve capacity as compared to the AD-N LCLs . Glycolytic reserve capacity changed significantly as DMNQ increased although this pattern of change was not significantly different across the two groups. Inhibition of UCP2 Affects AD-N and AD-A LCLs Differently In order to determine the differential ability of the AD LCL subgroups to adapt to intramitochondrial oxidative stress at the inner mitochondrial membrane, we used genipin to inhibit UCP2, the key protein on the inner mitochondrial membrane which regulates proton leak to reduce ETC generated oxidative stress. 8 Mitochondrial Dysfunction in Autism Cell Lines For this set of experiments, we used only two concentrations of DMNQ, 0 mM and 10 mM. Overall, LCLs exposed to genipin exhibited higher ATP-linked respiration than unexposed LCLs . ATP-linked respiration was also overall higher for the AD-A than the AD-N LCLs. Interestingly, there was a DMNQ by genipin interaction 15130089 such that ATP-linked respiration did not increase with DMNQ for the LCLs unexposed to genipin, but it increased significantly with DMNQ in the LCLs exposed to genipin. Overall, proton leak respiration was greater for the LCLs exposed to genipin as compared to the unexposed LCLs . It was also higher in the AD-A LCLs compared to the AD-N LCLs and in the LCLs exposed to DMNQ compared to the LCLs not treated with DMNQ. There was a DMNQ by 1417961 genipin interaction because the increase in proton leak respiration associated with DMNQ was greater for the LCLs exposed to genipin as compared to those unexposed to genipin. Genipin also resulted in a more significant increase in proton leak respiration for the AD-N LCLs as