Fig. 3: The LOAD-risk allele of PICALM impairs MG phagocytosis.

a, iMGs carrying LOAD-risk allele of PICALM show reduced phagocytosis of Aβ–pHrodo (compared with non-risk-allele iMGs), which can be rescued by PICALM-CRISPRa. The normalized Aβ–pHrodo intensity per iMG over a period of 180 min for both the CD04 and CD09 donor lines is shown. For each condition (genotype), data at each assay timepoint are from two independent experiments each with three single-well measurements (that is, differentiations). One clone per line. b,c, Representative western blot image (b) and quantification (c) of PICALM protein expression in iMGs of non-risk, risk and risk-CRISPRa for both lines. Protein quantity was normalized to β-actin. Gel source data are provided in Supplementary Fig. 1. Each data point represents a measurement of cell lysates from 12 wells of cultures from 1 experiment; for each donor line (CD04 and CD09) and each condition (non-risk, risk and risk-CRISPRa), data were collected from 3 independent experiments. One clone per line. d,e, PICALM KO by CRISPR–Cas9 editing C20 cells. The gRNA targeted at exon 1 depleted PICALM expression in immunoblot (d) and immunofluorescence labelling (e). Three experiments were performed. Scale bar, 50 μm. Gel source data are provided in Supplementary Fig. 1. f,g, Representative images (f) and quantification (g) of the phagocytosis of pHrodo-conjugated myelin in PICALM-KO C20 and control cells. Scale bar, 50 μm. In g, each data point represents a single-well measurement from one experiment (n = 4 wells in each group). Three experiments were performed with similar results. Cytochalasin D (10 µM) was used to show the specificity of phagocytosis. For all comparisons, analysis was performed using one-way analysis of variance (ANOVA) with Dunnett’s correction with the experimental round as the random factor. *P < 0.05; **P < 0.01; ***P < 0.001. Data are mean ± s.e.m.