Figure 4.

Teriflunomide increases glycolysis in quiescent astrocytes. Astrocytes grown under serum-free conditions were treated with teriflunomide (1, 10, 30 μM) under two paradigms. In the first, cells were incubated for 24 h. In the second, cells were treated once, then again after 48 h, and analyzed 24 h later (72 h total). Mitochondrial ATP production rate (A), glycolytic ATP production rate (B), and total ATP production rate (C) were measured using the Seahorse XF analyzer. Data are normalized to total protein (μg) per well. Each symbol represents one well from 3 separate experiments, shown in nested layout. Mean ± SEM is shown for each biological replicate. P-values were calculated using mixed model nested ANOVA between treatment conditions. (D) The ratio of mitochondrial ATP production rate to glycolytic ATP production rate was calculated from the data in (A) and (B) to indicate the ATP rate index in the 72 h paradigm. Each symbol represents one well from 3 separate experiments, shown in nested layout. Mean ± SEM is shown for each biological replicate. P-values were calculated using mixed model nested ANOVA between treatment conditions. (E) Viability of astrocytes exposed to teriflunomide (30 μM) in the 72 h paradigm was measured by MTT assay. Data are normalized to vehicle-only. Each symbol represents one well from 3 separate experiments, shown in nested layout. Mean ± SEM is shown for each biological replicate.