Figure 1.

ATM-knockout (KO) microglia are activated, resulting in increased expression of pro-inflammatory cytokines and enhanced phagocytic clearance. (A) Representative immunoblot analysis of WT, ATM KO and ATM KO cells in which ATM was re-expressed from a piggyBac transgene. To test for ATM activity, cells were treated with 1 μM camptothecin (CPT) for 1 h. Loading control: GAPDH. (B) Cell surface expression levels of the microglial activation marker, CD40, measured by flow cytometry in cells as in (A). Expression is relative to WT HMC3. Positive control: 10 μg/ml TNFα for 6 h, cells collected for analysis at 20 h post-treatment. Mean ± SD shown (n = 4). (C) RT-qPCR analysis of the indicated cytokines in cells as in (A). Expression is relative to WT HMC3 (dashed line). Reference genes: RSP13 and IPO8. Mean ± S.D. shown (n = 3). One-way ANOVA with Dunnett's multiple comparison's test used. (D) Representative immunofluorescence images of phagocytic uptake of 5 μm beads in WT and ATM KO HMC3 (6 h assay). Confocal Z-stack compression images shown. Arrowheads indicate disruption in vinculin structure associated with bead engulfment. Scale bar: 50 μm. Bead substrates (green), vinculin (red), DNA (blue). (E) Kinetics of phagocytosis (5 μm beads) in WT and ATM KO cells at the indicated time points. Phagocytosis is relative to WT HMC3 at 6 h with 3.7 ± 1.3% of phagocytic cells. Mean ± S.D. shown (n = 3). (F) Kinetics of the changes in percentage of highly phagocytic cells (>2 beads per cell) as in (E). To determine the number of beads per cell, Mean fluorescence intensity (MFI) of the population of phagocytic cells was divided by MFI of a single 5 μm fluorescent bead. Mean ± S.D. shown (n = 3). Unpaired two-tailed t-test used. (G) Phagocytosis levels (6 h assay) of cells as in (A). Phagocytosis is relative to WT HMC3, in which 5.8 ± 3.2% of cells are phagocytic. Mean ± S.D. shown (n = 4). (B, E, G) One-way ANOVA with Tukey's multiple comparison's test used.