Volume 20, Issue 5, Pages 1050-1060 (August 2017) Intrinsic Autophagy Is Required for the Maintenance of Intestinal Stem Cells and for Irradiation-Induced Intestinal Regeneration  Jumpei Asano, Taku Sato, Shizuko Ichinose, Mihoko Kajita, Nobuyuki Onai, Shigeomi Shimizu, Toshiaki Ohteki  Cell Reports  Volume 20, Issue 5, Pages 1050-1060 (August 2017) DOI: 10.1016/j.celrep.2017.07.019 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 20, 1050-1060DOI: (10.1016/j.celrep.2017.07.019) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Reduced Numbers of ISCs in Atg5ΔIEC Mice (A) Sections were prepared from the jejunum of GFP-LC3 tg mice (left panel) and Atg5ΔIEC GFP-LC3 tg mice (right panel). GFP-LC3 dots were observed by confocal laser microscopy. Staining for nucleus with DAPI in iECs is shown in blue. GFP-LC3 dots were detected in the crypts under steady-state conditions. Data are representative of five independent experiments. Original magnification, 60×. Scale bar, 10 μm. (B) p62 expression in Paneth cells and ISCs of control mice and Atg5ΔIEC mice was estimated by ELISA. Paneth cells and ISCs were sorted from the small intestine of control Lgr5 and Atg5ΔIEC Lgr5 mice. Data are representative of three independent experiments. ∗p < 0.04, ∗∗p < 0.01. (C) Representative flow cytometry (FCM) profiles of crypt cells from the jejunum of control and Atg5ΔIEC mice. The cells were divided into four major subpopulations based on the expression of c-Kit and CD24: goblet cells (c-Kit+CD24−, a), Paneth cells (c-KithighCD24high, b), progenitors (c-Kit−CD24low, c), and enteroendocrine cells (c-Kit−CD24high, d). (D) Percentage of each subpopulation. Data represent the means ± SD; n = 3; ∗p < 0.02. (E) Representative FCM profiles of ISCs from the jejunum of control Lgr5 and Atg5ΔIEC Lgr5 mice. (F) Percentage of ISCs and TA cells. Data represent the means ± SD; n = 5; ∗p < 0.005. Cell Reports 2017 20, 1050-1060DOI: (10.1016/j.celrep.2017.07.019) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Atg5ΔIEC Mice Show Defective Irradiation-Induced Intestinal Regeneration (A) H&E staining of the jejunum before irradiation and 24 hr, 5 days, and 7 days after irradiation. Data are representative of five independent experiments. Original magnification, 20×. Scale bar, 100 μm. (B) Ki-67 staining of cross-cut sections of the jejunum before irradiation and 24 hr, 5 days, and 7 days after irradiation. Data are representative of three independent experiments. Original magnification, 4×. Scale bar, 300 μm. (C) Number of crypts containing more than ten Ki-67+ cells per section of the indicated strains. Data represent the means ± SD; n = 3; ∗p < 0.03, ∗∗p < 0.005. (D) Crypts isolated from the jejunum of control and Atg5ΔIEC mice were cultured in Matrigel plus Advanced DMEM/F12 for 7 days. A representative organoid on day 7 is shown. Original magnification, 4×. Scale bar, 300 μm. (E) Organoid-formation efficiency (%) = ratio of the number of organoids detected on day 7 to the number of crypts seeded on day 0. Data represent the means ± SD; n = 3; ∗p < 0.005. Cell Reports 2017 20, 1050-1060DOI: (10.1016/j.celrep.2017.07.019) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Intrinsic Autophagy Deficiency in ISCs Causes ISC Reduction (A) Representative FCM profiles of ISCs from the small intestine of control Lgr5 and Ah-Cre: Atg5fl/fl Lgr5 mice, before irradiation and 24 hr and 7 days after irradiation. All mice were injected with βNF before starting the experiment. (B) Percentage of ISCs in the small intestine of control Lgr5 and Ah-Cre: Atg5fl/fl Lgr5 mice, before irradiation and 24 hr and 7 days after irradiation. Data represent the means ± SD; n = 3; ∗p < 0.01, ∗∗p < 0.005. (C) Atg5 mRNA level in purified ISCs from the small intestine of control Lgr5 and Ah-Cre: Atg5fl/fl Lgr5 mice 2 weeks after βNF injection. Data represent the means ± SD; n = 3; ∗p < 0.002. (D) Atg5 protein expression in purified ISCs from the small intestine of the indicated strains 2 weeks after βNF injection, analyzed by western blot. Bands representing the ATG5-ATG12 complex and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (loading control) are shown. Data are representative of two independent experiments. (E) H&E staining of the jejunum before irradiation and 24 hr and 7 days after irradiation. Data are representative of three independent experiments; n = 3. Original magnification, 20×. Scale bar, 100 μm. Cell Reports 2017 20, 1050-1060DOI: (10.1016/j.celrep.2017.07.019) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Levels of Mitochondria and ROS in Atg5ΔIEC IEC Subpopulations (A) The number of mitochondria in the progenitors was evaluated by Mitotracker Green FM. Bold solid lines represent cells stained with Mitotracker Green FM, and thin solid lines show unstained controls (blue, control mice; red, Atg5ΔIEC mice). Data are representative of three experiments. (B) The number of mitochondria in each subpopulation. ΔMFI = MFI of Mitotracker Green FM staining − MFI of the unstained control. Data represent the means ± SD; n = 3; ∗p < 0.01. (C and D) Mitochondrial membrane potential and ROS amount in progenitors, evaluated by Rh123 (C) and CellROX (D), respectively. Bold solid lines represent cells stained with Rh123 and CellROX, and thin solid lines show unstained controls of the indicated strains. Data are representative of three independent experiments (left panels). Right panels show the percentage of low-peak mitochondrial membrane potential and the amount of ROS in the progenitors of the indicated strains. ΔMFI = MFI of CellROX staining − MFI of unstained control. Data represent the means ± SD; n = 3 (right panels); ∗p < 0.02 (C), ∗p < 0.05 (D). (E and F) ROS levels in ISCs and TA cells. Data are representative of three independent experiments (E) and show the means ± SD; n = 5; ∗p < 0.04 (F). Cell Reports 2017 20, 1050-1060DOI: (10.1016/j.celrep.2017.07.019) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 ISC Homeostasis Is Restored by Limiting the Increase in ROS (A and B) Crypts isolated from the jejunum of control and Atg5ΔIEC mice were cultured for 7 days in the absence or presence of NAC. (A) Representative image of an organoid on day 7. Original magnification, 4×. Scale bar, 300 μm. (B) Organoid-formation efficiency (%). Data represent the means ± SD; n = 3; ∗p < 0.02, ∗∗p < 0.05. (C) BrdU staining of the jejunum crypts of control and Atg5ΔIEC mice before and 3.5 days after irradiation. Some mice were treated with NAC (NAC+) before irradiation. Original magnification, 100×. Scale bar, 20 μm. (D) Number of BrdU+ cells per crypt (100 crypts were analyzed) of the jejunum of control and Atg5ΔIEC mice. Data represent the means ± SD; n = 3; ∗p < 0.01, ∗∗p < 0.03. (E) Representative FCM profiles of ISCs from the jejunum of control and Atg5ΔIEC Lgr5 mice 7 days after irradiation. (F) Percentage of ISCs and TA cells. Data represent the means ± SD; n = 3; ∗p < 0.01, ∗∗p < 0.02. Cell Reports 2017 20, 1050-1060DOI: (10.1016/j.celrep.2017.07.019) Copyright © 2017 The Author(s) Terms and Conditions