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. 2022 Oct 6;13:5688. doi: 10.1038/s41467-022-33364-z

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© The Author(s) 2022

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. 10 — a, b Generation of CRISPR/Cas9-engineered PSC lines and organoids with homozygous and complete hemizygous SHANK3 deletions (SHANK3-/- and SHANK3+/−, respectively) (elements were drawn by Coni Hoerndli Science Design). c Images of 5-month-old iCtrl and SHANK3+/− SNR-derived organoids. d Synaptic stainings using anti-SYN1 and anti-SHANK3 antibodies in iCtrl and SHANK3-deficient organoids (left: iCtrl [2242-5] and SHANK3-/- [EP2-15]; right: iCtrl [H9] and SHANK3+/− [EYQ]). e Quantification of SYN1/SHANK3 puncta (top: n = 7 and 12 sections from 2 iCtrl [2242-5] and 3 SHANK3−/− [EP2-15] organoids produced in two batches, respectively, ***P = 0.0007, unpaired two-sided Mann–Whitney test; bottom: n = 11 and 11 sections from 3 iCtrl [H9] and 3 SHANK3+/−[EYQ] organoids produced in two batches, respectively, *P = 0.029, unpaired two-sided Mann–Whitney test). f Traces of voltage deflections recorded from iCtrl (2242-5) and SHANK3-/- (EP2-15) neurons in response to different somatic current injections (ΔI = 10pA). g Quantified Rheobase AP latency, membrane capacitance, AP threshold, AHP, and AP firing rate for iCtrl and SHANK3-deficient neurons in 5-month-old SNR-derived organoid slices (n = 32, 18, 36, and 17 cells, from 4 iCtrl [2242-5], 3 SHANK3-/- [EP2-15], 11 iCtrl [H9], and 2 SHANK3+/− [EYQ] organoids, [P = 0.053 and 0.039 [Rheobase AP latency], 0.012 [membrane capacitance], 0.038 [AP threshold], 0.011 [AHP], 0.041 [AP firing rate], unpaired two-sided t-test). These cells are also included in analyses presented in Fig. 8i and 9d. h UMAP visualizations of unsupervised clustering of scRNA-seq data from 2 iCtrl [H9] and 2 SHANK3+/− [EYQ] SNR-derived organoids and relative cluster composition (right). These organoids are also included in the clustering results presented in Fig. 5. i Heatmap of expression of different cell-type specific marker genes in iCtrl (H9) and SHANK3+/− (EYQ) organoids. j, k Volcano plot visualizations of DEGs from bulk RNA-seq data obtained from 3 iCtrl (H9) and 3 SHANK3+/− (EYQ) organoids (j) and 6 control and 6 SHANK3+/− organoids. Gene ontology (GO) analysis performed on most downregulated genes (Log2 fold change < −1.5) detected in SHANK3+/− organoids. l Relative expression of clustered protocadherins in 6 control and 6 SHANK3+/− organoids (P = 0.0022 [PCDHA6], 0.0022 [PCDHA7], 0.009 [PCDHA9], 0.009 [PCDHB7], 0.041 [PCDHB8], 0.0022 [PCDHGA1], 0.0022 [PCDHGA2], and 0.0043 [PCDHGB5], unpaired two-sided Mann–Whitney test). Data are mean ± s.e.m. Scale bars = 450 (c), and 50, 20, and 5 μm (d). Source data are provided as a Source Data file.