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. 2016 Feb 29;212(5):545–560. doi: 10.1083/jcb.201507023

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© 2016 Wang et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 1. — Atg5 and Atg16L1 deletion causes deficient autophagy and mitochondria accumulation in postnatal NSCs. (A) Lysates were extracted from the SVZ of P14 Ctrl, Atg5^GFAP cKO, and Atg16L1^GFAP cKO mice treated with chloroquine for 7 d and analyzed by immunoblot to detect Atg5 (left), Atg16L1 (right), LC3, p62, and Vinculin. The asterisk on the right marks a band detected by anti-Atg16L1 antibody either nonspecifically or as a Atg16L1 degradation product in some samples. (B and C) Atg5, Atg16L1, and DAPI immunofluorescence in the SVZ (B; n = 3 mice each) and neurospheres (C) from Ctrl, Atg5^GFAP cKO, and Atg16L1^GFAP cKO SVZ cells at P28. (D) LC3 and DAPI immunofluorescence in neurospheres from Ctrl, Atg5^GFAP cKO, and Atg16L1^GFAP cKO SVZ cells at P28. Boxed areas show more detail for LC3 puncta. LC3 puncta number per nuclei (mean ± SEM) are graphed on the right (n = 3 mice each, >300 cells from three mice). (E) TEM of mitochondria (examples marked by arrows) in Ctrl, Atg5^GFAP cKO and Atg16L1^GFAP cKO SVZ tissues at P28. Mitochondria number per nuclei (mean ± SEM; n = 2 mice each, >30 cells from each mouse). (F) Total (MitoTracker Green) and functional (MitoTracker Red) mitochondria by FACS analysis of dissociated neurospheres cells from Ctrl, Atg5^GFAP cKO, and Atg16L1^GFAP cKO SVZ at P28 (n = 3 mice each). (G) Dysfunctional mitochondria were evaluated by the percentage of cells with decreased ratios of MitoTracker Red to MitoTracker Green (n = 2 mice each). E, ependymal layer; LV, lateral ventricle; N, nuclei; ST, striatum. Bars: (B and C) 30 µm; (D and insets) 6 µm; (E) 1 µm. **, P < 0.01; ***, P < 0.001.