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. 2022 Jan 8;10:4. doi: 10.1186/s40478-021-01285-5

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© The Author(s) 2022

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 8 — Rescue of endosomal phenotype in BIN1 null mutant neurons by transduction of BIN1 Isoform 1. a–a’’’ Representative images of BIN1 WT and KO hiNs in 6-weeks-old 2D culture immunolabeled with antibodies against tdTomato (tdT, red, a), EEA1 (green, a’), MAP2 (grey, a’’), and stained with Hoechst 33258 (blue, a’’’) (Scale Bar = 10 µm). b Plot showing the cumulative distribution of EEA1⁺ puncta volumes in transduced BIN1 KO hiNs with tdT lentiviral constructs; BIN1iso1 (dashed red line), BIN1iso9 (dashed red line), and Mock-tdT (solid red line). BIN1 WT hiNs tranduced with Mock-tdT (solid blue line) is indicated to show the rescue effect of the BIN1iso1 construct (orange line) in BIN1-null mutant neurons. Kolmogorov–Smirnov test followed by Bonferroni correction: BIN1 WT + Mock vs BIN1 KO + Mock: ****Padj < 0.0001; BIN1 WT + Mock vs BIN1 KO + iso9: Padj < 0.0001; BIN1 KO + Mock vs BIN1 KO + Iso1: ****Padj < 0.0001; BIN1 WT + Mock vs BIN1 KO + Iso1: Padj = 0.174; BIN1 KO + Mock vs BIN1 KO + Iso9: Padj = 0.207 (N = 3 independent cell cultures). c Plot showing the cumulative distribution of EEA1⁺ puncta volumes in transduced BIN1 WT hiNs with tdTomato (tdT)-expressing lentiviral constructs; BIN1iso1 (dashed blue line), BIN1iso9 (dotted blue line), and Mock-tdT (solid blue line). Kolmogorov–Smirnov test followed by Bonferroni correction: BIN1 WT + Mock vs BIN1 WT + Iso1: *Padj = 0.04; BIN1 WT + Mock vs BIN1 WT + Iso9: ****Padj < 0.0001 (N = 3 independent cell cultures). d Graph showing the quantification of numbers of EEA1 puncta per cell in cells transduced with tdT-tagged lentiviral constructs (N = 3 independent cell cultures. EEA1 ⁺ puncta/neuron: BIN1 WT + Mock: 65.86 ± 12.78; BIN1 WT + Iso1: 59.75 ± 8.895; BIN1 WT + Iso9: 69.82 ± 11.42; BIN1 KO + Mock: 56.47 ± 12.27; BIN1 KO + Iso1: 55.65 ± 14.52; BIN1 KO + Iso9: 77.31 ± 11.04; p = 0.81, ANOVA _F(5,90)). e Quantification of MAP2⁺ /tdTomato⁺ neurons in BIN1iso1- and BIN1iso9-transduced cells relative to Mock-tdT-transduced cells (N = 3 independent cell cultures; Number of tdT⁺ cells: Mock = 468; BIN1iso1 = 193; BIN1iso9 = 103; ANOVA followed by Dunnett’s multiple comparisons test, **p = 0.0289)