Figure 8.

PDK3 KD decreases PDH phosphorylation and improves mitochondrial respiration. Striatal cells were transfected with plko.1-shRNA against PDK3 or plko.1-scramble vector 48 h before the experiments or incubated for 24 h with 500 μm SB. The efficacy of the KD was confirmed by Western blotting probed for PDK3 (A) and by decreased PDH E1alpha subunit phosphorylation at Ser293 and Ser300 (B). Statistical analysis was performed by two-way ANOVA and a significant interaction between genotype and PDK3KD was found (F_{(2, 40)} = 3.675, p = 0.0343). In addition, PDK3KD had a significant effect on Ser293 (F_{(2, 40)} = 7.693, p = 0.0015) and Ser300 (F_{(2, 40)} = 27.62, p < 0.0001) phosphorylation. The OCR was measured with a Seahorse Bioscience XF24 flux analyzer (C). Basal mitochondrial respiration is represented in Ci and exhibits a significant interaction between genotype and treatment (F_{(3, 25)} = 3.248, p = 0.0387). A strong, significant effect of treatment on basal respiration was also observed (F_{(3, 25)} = 12.57, p < 0.0001). Striatal cells were treated for the indicated times with 1 μm oligomycin A, 0.9 μm FCCP, and 1 μm antimycin A plus 1 μm rotenone to determine the proportion of oxygen consumption due to ATP turnover (Ciii) and maximal rate of respiration (Cii). PDK3KD or SB treatment had a significant effect on both Cii (F_{(3, 26)} = 4.817, p = 0.0085) and Ciii (F_{(3, 26)} = 4.436, p = 0.0120). Results are expressed as mean ± SEM of three to four independent experiments. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared with untransfected cells; ^####p < 0.0001 compared with mutant untransfected cells; ^$p < 0.05, ^$$p < 0.01, and ^$$$$p < 0.0001 compared with plko.1-transfected cells by two-way ANOVA followed by Bonferroni post test; ^tttp < 0.001 compared with the wild-type (Wt) control (in Cii) by Student's t test.