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. 2020 Jul 7;26(10):5733–5750. doi: 10.1038/s41380-020-0807-4

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — PITRM1^+/+ and PITRM1⁻^/− cerebral organoids were treated with 500 nM ISRIB or vehicle, daily, from DIV 20 to DIV 50. a, b MAP2 (green), APP (red, in a), and phospho-tau (p-tau; red, in b) immunostaining in PITRM1^+/+ and PITRM1⁻^/− cerebral organoids treated with ISRIB or vehicle. Representative confocal images are shown. Cell nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. c Quantification of APP and phospho-tau mean fluorescent intensity (mean + SEM; *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed t test, n = 3–4). d Quantification of Aβ species in the supernatant of PITRM1^+/+ and PITRM1⁻^/− cerebral organoids treated with ISRIB or vehicle, as measured by Meso Scale immunoassay (mean + SEM; *p < 0.05, ***p < 0.001, two-tailed t test, n = 6–7). e Immunostaining for β-TUBIII (green) and cleaved caspase-3 (cCASP3, red) in PITRM1^+/+ and PITRM1⁻^/− cerebral organoids treated with ISRIB or vehicle. Cell nuclei were counterstained with DAPI (blue). Scale bar, 100 µm. Quantification of the ratio of cCASP3-positive cells relative to the total number of β-TUBIII/DAPI cells is shown (mean + SEM; **p < 0.01, two-tailed t test, n = 3–4). f mtDNA content was measured in PITRM1^+/+ and PITRM1⁻^/− cerebral organoids treated with ISRIB or vehicle as the mitochondrial (16S) to nuclear (RPLP0) DNA ratio by qRT-PCR (mean + SEM; *p < 0.05, two-tailed t test, n = 4). g–j PITRM1^+/+ and PITRM1⁻^/− cerebral organoids were treated with 500 μM NMN or vehicle, daily, from DIV 45 to DIV 50. g mtDNA content was measured as the mitochondrial (16S) to nuclear (RPLP0) DNA ratio by qRT-PCR (mean + SEM; **p < 0.01, ***p < 0.0001, two-tailed t test, n = 3). h Aβ1–42/Aβ1–40 ratio in cerebral organoids measured by Meso Scale immunoassay (mean + SEM; **p < 0.01, two-tailed t test, n = 4–7). i ELISA assay measuring the levels of total and phospho-tau levels in cerebral organoid homogenates. The results are presented as the ratio of phospho-tau/total-tau (mean + SEM; ***p < 0.0001, two-tailed t test, n = 4). j Immunostaining and quantification for β-TUBIII (green) and cleaved caspase-3 (cCASP3, red) in PITRM1⁻^/− cerebral organoids treated with NMN or vehicle. Cell nuclei were counterstained with DAPI (blue). Scale bar, 100 µm. Analysis of the ratio of cCASP3-positive cells relative to the total number of β-TUBIII/DAPI cells is shown (mean + SEM; **p < 0.01, two-tailed t test, n = 4).