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. 2017 Mar 8;37(10):2776–2794. doi: 10.1523/JNEUROSCI.2006-14.2016

Figure 2.

Figure 2.

Protective effect of PDH modulators in striatal mutant cells. Cell viability (A–E) and redox signaling (F, G) of wild-type and mutant cells was analyzed 24 h after incubation with different concentrations of the PDH activator DCA (0.3–3 mm) or the acetyl-coenzyme A donor ALCAR (0.1–5 mm). The effects of these compounds were assessed on PDH activity (H) and their mechanisms of action are represented in I. A–E, To analyze cell viability, cells were stained with fluorescent Hoechst 33342 (blue) and propidium iodide (red) dyes and visualized by fluorescence microscopy as in the images shown in A to evaluate the nuclear morphology (DNA condensation and fragmentation) to assess apoptotic cell death (full arrows) and plasma membrane integrity to assess the number of necrotic cells (dashed arrows). Cell viability, necrosis, and apoptosis were expressed as a percentage of the total number of cells. In A–E, statistical analysis was performed by one-way ANOVA. F, G, To analyze altered redox signaling, cells were loaded with DCFH2-DA (20 μm) for 30 min 24 h after exposure to DCA or ALCAR and the fluorescence of DCF (480 nm excitation, 550 nm emission) resulting from the probe oxidation was followed continuously for 1 h at 33°C and corrected for total protein. Results are expressed as mean ± SEM of three experiments performed in triplicate or quadruplicate. The wild-type control (CTR) 100% of DCF fluorescence was 3.67 × 106 ± 0.7 × 106 RFU/min/mg protein. In F and G, statistical analysis was performed by two-way ANOVA, followed by Bonferroni post test, and revealed a significant interaction between genotype and treatment in F (F(3, 38) = 6.161, p < 0.0016) and a significant effect of ALCAR treatment in G (F(3, 40) = 3.558, p < 0.0225). H, PDH activity was measured by spectrophotometry. Results are expressed as mean ± SEM of three independent experiments (performed in triplicate) and wild-type values (100%) correspond to 1178.00 ± 247.00 AU/min/mg protein. Statistical significance: *p < 0.05 or ***p < 0.001 compared with the respective wild-type condition and ##p < 0.01 or ###p < 0.001 compared with control (untreated) condition. For H, tttp < 0.001 by the Student's t test.