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. Author manuscript; available in PMC: 2020 Jul 25.
Published in final edited form as: Cell. 2019 Jun 27;178(3):536–551.e14. doi: 10.1016/j.cell.2019.05.056

Figure 2. ATG5 and Rubicon-deficiency impairs LANDO and recycling of Aβ receptors.

Figure 2.

A. Representative images showing that GFP-LC3-recruitment to Aβ (red) containing vesicles in BV2 microglia is dependent on ATG5 and Rubicon, but not FIP200. White arrows indicate LC3+ endosomes. Cells were treated with 1μM oligomeric TAMRA-Aβ1-42 for 6h. Scale bars, 5μm. Values are (# of cells containing LC3+, Aβ+ vesicles/100 Aβ-containing cells).

B. Quantification of membrane-associated GFP-LC3 in BV2 microglia following 6h stimulation with 1μM oligomeric TAMRA-Aβ1-42. GFP-LC3 was assayed using flow cytometry. Each point represents one independent experiment performed in triplicate.

C. Quantification of zymosan (4:1, particle:cell), dextran (500ng/ml), or Aβ (1μM) uptake in BV2 microglia treated with either a vehicle or 50mM latrunculin A (LA). n=3 per condition performed in duplicate.

D. Aβ clearance assay performed in BV2 microglia treated with oligomeric TAMRA-Aβ1-42 for 1h. n=4 per genotype performed in duplicate.

E. Quantification of fluorescent pH-rodo signal from BV2 microglia of the indicated genotypes stimulated with pH-rodo Zymosan (5:1, particle:cell) or pH-rodo Aβ (1μM) for 3h. n=3 per genotype performed in duplicate.

F. Quantification of the co-localization between zymosan or Aβ and LAMP1 labeled lysosomes in BV2 microglia (see Fig. S2F). Cells were treated with zymosan (4:1, particle:cell), or Aβ (1μM) for 3h. n=3 per genotype performed in duplicate.

G. Primary and secondary uptake of Aβ measured in BV2 microglia. Each point represents one independent experiment performed in duplicate.

H. Representative images of receptor recycling for TLR4, TREM2, and CD36 in BV2 microglia. Scale bars, 50mm.

I. Quantification of recycled receptors in BV2 microglia (See STAR Methods). Each point is one independent experiment performed in duplicate.

J. Representative images of TREM2 recycling in primary microglia from Rubicon+/− or Rubicon−/− mice. Scale bars, 10μm.

K. Quantification of TREM2 recycling in primary microglia from indicated genotypes. Each point is one independent experiment performed in duplicate.

Data are represented as mean ± SEM. Significance was calculated using Student’s t-test. *p<0.05, **p<0.01, ***p<0.001.