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. Author manuscript; available in PMC: 2016 Nov 23.
Published in final edited form as: Science. 2016 Mar 18;351(6279):1324–1329. doi: 10.1126/science.aaf1064

Fig. 3. Analysis of macrophages and microglia from C9orf72 deficient mice.

Fig. 3

(A) qRT-PCR analysis from B cells, T cells, and CD11b+ cells FAC sorted from wild-type mouse spleen (n=2). (B,C) Bone marrow derived macrophages (BMDMs) from C9orf72−/− mice showed accumulation of LysoTracker and Lamp1 stained vesicles compared to wild-type (Wt). Scale bar = 50 µm and 20 µm. (D) C9orf72−/− BMDMs treated with lentivirus encoding either human C9orf72 isoform 1-IRES-GFP (hC9-iso1) or isoform 2-IRES-GFP (hC9-iso2). LysoTracker (top panel) or Lamp1(bottom panel) accumulation was rescued by either hC9-iso1 or hC9-iso2 (top panel). Arrow: hC9-iso1 infected cell; asterisk: uninfected cell. (E) Quantitation of LysoTracker accumulation in BMDMs of the indicated genotype, or homozygotes treated with hC9-iso1 and hC9-iso2 lentivirus. (***p=0.0002, **p=0.0018, one-way ANOVA). (F) BMDMs fed with fluorescent zymosan particles for 15 minutes and then analyzed by FACS analysis. (G) ROS production by BMDMs after zymosan ingestion in indicated genotypes (****p=<0.0001, two way ANOVA). (H) C9orf72+/− and C9orf72−/− BMDMs showed increased TNFα production after stimulation with Pam3CSK4 (Pam), peptidoglycan (PGN) and CpG, but not lipopolysaccharide (LPS) (****p<0.0001, ***p=0.0002, two way ANOVA. N.D. – not detected). (I) IL-1 beta production after stimulation with silica (*p<0.05, two-way ANOVA). (J) RNA-seq of C9orf72 in indicated cell types from the cerebral cortex (21). (K) qRT-PCR of C9orf72 from neurons and microglia isolated from adult mouse brain. (L) Microglia purified from C9orf72−/− mice showed accumulation of LysoTracker and Lamp1 positive enlarged vesicles. (M) Quantification of percentage of microglia with enlarged LysoTracker positive vesicles (*p=0.027, one-tailed t-test).