Fig. 6

Iluzanebart (ILU) increases viability and restores CSF1R activity in CRISPR-generated I794T^+/-  mutant iPSC microglia. iPSC microglia generated to heterozygously express the I794T CSF1R mutation were harvested, plated onto IgG- or iluzanebart-coated surfaces (ILU), and incubated for 7 days before analysis. Cultures were assessed for viability by CellTiterGlo, soluble CSF1R in the media by ELISA, or for phospho-CSF1R levels in the cells by ELISA. A In I794T^+/- microglia, viability was significantly increased in iluzanebart-treated cultures compared to IgG-treated controls (n = 9 independent experiments). B While iluzanebart treatment did not significantly affect levels of total CSF1R in either WT or I794T^+/- cells, total CSF1R levels were 23% lower in I794T^+/- cells compared to WT cells (n = 9 independent experiments). C While soluble CSF1R levels were 38% lower in I794T^+/- cell culture media compared to WT, iluzanebart treatment significantly increased levels of soluble CSF1Rin both cultures (n = 9 independent experiments). D While iluzanebart treatment had no impact on phosphorylation of CSF1R in WT cells, phospho-CSF1R levels were 34% lower in I794T^+/- cells compared to WT, and iluzanebart treatment restored pCSF1R levels to normal WT levels. (n = 9 independent experiments). E While iluzanebart treatment slightly increased the ratio of activated CSF1R in WT cells, activated CSF1R levels were increased 2.26 × in I794T^+/- cell cultures (n = 9 independent experiments). All data were normalized to cell number as determined using Incucyte cell number determination. Significance was determined by two-way ANOVA with Tukey’s multiple comparison’s test, * < 0.05, ** < 0.005, **** < 0.0001