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. 2023 Jan 28;14:455. doi: 10.1038/s41467-023-36081-3

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© The Author(s) 2023

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. 8 — a–c Mouse hippocampal neuronal cultures were transfected as indicated and imaged at DIV14-17. a Representative confocal images (top), and corresponding line-scan analysis (bottom) of neuronal cultures expressing synaptophysin-HA with ATG9A-EGFP or mCherry-SCAMP5. Synaptophysin-HA was detected by anti-HA immunofluorescence. b Colocalization analysis calculated by Pearson’s coefficients. Values are means ± SEM. **p < 0.01 by the two-sided Student’s t-test (all boutons in 8 field of view (121 μm x 121 μm) for each condition from two independent cultures were quantified). c Localization of ATG9A-EGFP, synaptophysin-HA and mCherry-synapsin in presynaptic varicosities of an axon. Synaptophysin-HA was visualized by anti-HA immunofluorescence. d–g Mouse hippocampal neuronal cultures were fixed and stained as indicated at DIV14-17 to visualize endogenous localization of ATG9A in neurons. d, e Representative confocal images showing immunoreactivity for endogenous ATG9A, synaptophysin and VAMP2 (d) or Rab3A (e) in cultured hippocampal neurons. Corresponding line-scans are shown at the bottom. f, g Colocalization analysis. Values are means ± SD; N.S., not significant; **p < 0.01 by one-way ANOVA with the Tukey’s HSD post hoc test (12 images (121 μm x 121 μm for each) from two independent cultures were analyzed). Source data are provided as a Source Data file. Scale bars, a = 5 μm, c = 10 μm (1 μm for high-magnification images), d and e = 2 μm. p-values (b): 9 × 10^‒9, (f): 0 [(Syph vs. VAMP2) vs. (Syph vs. ATG9A)], 0 [(Syph vs. VAMP2) vs. (VAMP2 vs. ATG9A)], 0.8080 [(Syph vs. ATG9A) vs. (VAMP2 vs. ATG9A)]. (g): 0 [(Syph vs. Rab3A) vs. (Syph vs. ATG9A)], 0 [(Syph vs. Rab3A) vs. (Rab3A vs. ATG9A)], 1.826 [(Syph vs. ATG9A) vs. (Rab3A vs. ATG9A)].