Extended Data Fig. 2: ATV:4D9 induces temporally dynamic microglial morphology and marker expression.

(a) Representative morphometric images of microglia in the cortex 1 day post dose with ATV:ISO or ATV:4D9 (10 mg kg⁻¹) at day 1-, 7-, 14-, and 28-days post dose. (b) UMAP plot of all microglia over time. (c) Percentage of microglia in the responsive cluster as a proportion of all segmented microglia over time (n = 3 male mice per group, two-tailed unpaired t-test between ATV:ISO and ATV:4D9 at day 1, mean ± SEM). (d) Volcano plot showing the top 6 differentially high and low normalized features comparing the responsive cluster to the homeostatic cluster. (e) Heatmap of normalized features for all segmented microglia (1,143 total cells) over time, measured across 65 morphometric and immunohistochemical features, grouped by treatment (rows) with features hierarchically clustered (columns). (f) Representative images of cortical brain sections co-stained with Iba1 and CD74 at 1-, 7-, 14-, 28-days days post 10 mg kg⁻¹ dose of ATV:ISO or ATV:4D9. CD74⁺ microglia are noted with white arrows. (g) Mean intensity of CD74 staining within segmented IBA1⁺ microglia normalized to background CD74 intensity at each timepoint (n = 5 mice/group, two-tailed unpaired t-test between ATV:ISO and ATV:4D9 at day 1, mean ± SEM). (h) Representative images of cortical brain sections stained for IBA1 and AXL 1 day post dose of ATV:ISO or ATV:4D9. Double positive microglia are noted with white arrows. (i) Quantification of mean intensity of AXL staining within segmented IBA1⁺ microglia normalized to background (n = 5 mice/group, two-tailed unpaired t-test, mean ± SEM).