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. 2018 Jul 5;9:552. doi: 10.3389/fneur.2018.00552

Figure 6.

Figure 6

KL1333 increases the NAD+/NADH ratio and activates SIRT1, AMPK, and PGC-1α in MELAS fibroblasts. (A) MELAS 1 fibroblasts were treated with 1 μM KL1333 for 30 min lysed in extraction buffer, and intracellular ATP levels were measured. Intracellular NAD+ and NADH were extracted, and NAD+ and NADH levels were measured using a microplate reader. The NAD+/NADH ratio was calculated based on the concentration of NAD+ and NADH. (B) MELAS 1 fibroblasts were treated with 1 μM KL1333 for 30 min. SIRT1 activity was analyzed using a fluorescence-based assay. (C) MELAS 1 fibroblasts were treated with 1 μM KL1333 for the indicated times. The activity of AMPK was examined by Western blotting using the indicated antibodies. (D) Quantification of pAMPK levels in MELAS 1 fibroblasts using its band density normalized to that of total AMPK. (E) MELAS 1 fibroblasts were treated with 1 μM KL1333 for 24 h. The expression of PGC-1α and its target genes were measured by real-time PCR using specific primers for Ppargc1a, Tfam, Nrf1, Nrf2, and Sod2. Each experiment was repeated three times. Error bars indicate ±SEM. *P < 0.05.