Fig. 7. Pharmacological activation of autophagy with rapamycin ameliorates the sustained immune activation and phagocytic deficit in mC9-MG and C9KO-MG.

(A) Schematic showing the experimental setup wherein the cells are treated with rapamycin for 12 hours and tested for cytokine release and phagocytosis of zymosan bioparticles. (B to G) Cytokine ELISA of IL-6 and IL-1β demonstrates suppression of the production of proinflammatory cytokines in mC9-MG following rapamycin treatment. Cells were treated with either vehicle (veh), LPS, or LPS + rapamycin (LPS + Rap). Data are represented as means ± SD; statistical analysis was performed using two-way ANOVA and Sidak’s multiple comparisons test (*P ≤ 0.05); N = 3. (H and I) IL-1β and IL-6 ELISA demonstrates the amelioration of immune response in C9KO-MGs mirroring mC9-MGs as a result of rapamycin treatment; cells were treated with either vehicle (veh), LPS, or LPS + rapamycin. Data are represented as means ± SD; statistical analysis was performed using two-way ANOVA and Sidak’s multiple comparisons test. (**P < 0.01 and ***P < 0.001); N = 3. (J to M) Graph showing real-time imaging of zymosan bioparticle uptake assay demonstrating rapamycin-mediated amelioration of the phagocytic deficit in mC9-MGs and C9KO-MG; statistical analysis was performed across rapamycin-treated and rapamycin-untreated condition for all genotypes using two-way ANOVA and Tukey’s multiple comparisons test (*P <0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001) and error bars represent ± SEM; N = 3. N represents the number of times experiments were performed using cells generated from independent differentiations from iPSCs.