Fig. 2. mC9-MGs display impaired phagocytosis and heightened immune response following LPS stimulation.

(A) Schematic overview of the experimental paradigm for the assessment of microglial function—phagocytosis and immune response following LPS stimulation. ELISA, enzyme-linked immunosorbent assay. (B) Representative images of immunofluorescence staining showing phagocytosis assay performed with pH-sensitive zymosan bioparticles for three pairs of mC9-MG and isoC9-MG at 60 and 120 min [IBA-1 (green), zymosan bioparticles (red)]. Scale bars, 50 μm. (C) Graphs showing real-time imaging of zymosan bioparticle uptake at 15-min intervals, demonstrating a phagocytic deficit in mC9-MG when compared to isoC9-MGs across three pairs. Statistical analysis was performed across mC9-MG and isoC9-MG using two-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test; data are represented as means ± SEM, N = 3 (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). (D to F) Graphs showing increased production of IL-6 and IL-1β in mC9-MG (red) compared to isoC9-MG (blue) following LPS stimulation, cells were treated with either LPS or 1× phosphate-buffered saline (PBS) [vehicle control (veh)]. Statistical analysis was performed using two-way ANOVA and Tukey’s multiple comparisons test, data are represented as means ± SD; N = 3 (*P < 0.05;, **P < 0.01, and ***P < 0.001). (G to J) Immunoblot with respective densitometric analysis showing the reduced abundance of C9ORF72 protein (~55 kDa) across three lines of mC9-MG (red) when compared to their respective isogenics (blue). Data are represented as means ± SD; N = 3. Statistical analysis was performed using Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001). N across all experiments represents the number of times experiments were performed using cells generated from independent iPSC differentiations. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.