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. 2021 Jul 20;12:4399. doi: 10.1038/s41467-021-24490-1

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© The Author(s) 2021

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. 3 — a Representative EJC (above) and mEJC (below) traces, quantification of EJC amplitude, mEJC amplitude and frequency of control [UAS > GFP/+; HB9 > Gal4, Tub > Gal80^ts] and HB9 neuron selective adult conditional vglut^−/− mutants [Df(2R)371/B3RT_vglut_B3RT; HB9 > Gal4, UAS > GFP, Tub > Gal80^ts/UAS > B3] at 20 days after eclosion. b Representative images of presynaptic HB9 neuron boutons (red, GFP) and active zones (Brp, green), quantification of bouton diameters and percentage of boutons with only a single active zone (bouton fragmentation) of control and vglut mutants until 35 days after eclosion. c Representative EJC and mEJC traces, quantification of EJC amplitude, mEJC amplitude and frequency of control [UAS > luciferase^RNAi/HB9 > Gal4, UAS > GFP, Tub > Gal80^ts] and HB9 neuron selective adult conditional Para knockdown mutants (Para^KD) [UAS > GFP/+; HB9 > Gal4, Tub > Gal80^ts/UAS > Para^RNAi] at 20 days after eclosion. d Representative images of presynaptic HB9 neuron boutons and active zones, quantification of bouton diameters and bouton fragmentation as measured by the percentage of boutons with only a single active zone of control and Para^KD mutants until 35 days after eclosion. e Representative EJC and mEJC traces, quantification of EJC amplitude, mEJC amplitude and frequency of HB9 neuron selective adult conditional V100^WT [UAS > V100^3′UTR_RNAi/UAS > V100^WT; HB9 > Gal4, UAS > GFP, TubGal80^ts] and V100^WFI mutants [UAS > V100³^′^UTR_RNAi/UAS > V100^WFI; HB9 > Gal4, UAS > GFP, TubGal80^ts] at 20 days after eclosion. f Representative images of presynaptic HB9 neuron boutons and active zones, quantification of bouton diameters and percentage of boutons with only a single active zone of V100^WT and V100^WFI mutants until 35 days after eclosion. Unpaired two-tailed t-test, ***p < 0.001, n ≥ 9 presynaptic terminals per timepoint (a, EJC amplitude), p = 0.249, n ≥ 17 presynaptic terminals per timepoint (a, mEJC amplitude), ***p < 0.001, n ≥ 17 presynaptic terminals per timepoint (a, mEJC frequency), ***p < 0.001, n ≥ 6 presynaptic terminals per timepoint (c, EJC amplitude), p = 0.09, n ≥ 10 presynaptic terminals per timepoint (c, mEJC amplitude), p = 0.5, n ≥ 10 presynaptic terminals per timepoint (c, mEJC frequency), p = 0.4, n ≥ 8 presynaptic terminals per timepoint (e, EJC amplitude), p = 0.06, n ≥ 8 presynaptic terminals per timepoint (e, mEJC amplitude), **p = 0.004, n ≥ 8 presynaptic terminals per timepoint (e, mEJC frequency). Two-way ANOVA, followed by Sidak’s multiple comparison tests were used to compare the mean of control and experimental genotypes in that timepoint, ns = not significant, ***p ≤ 0.001, n = 150 boutons per timepoint (b, bouton diameters), ns not significant, ***p < 0.001, n ≥ 9 presynaptic terminals per timepoint (b, single active zone boutons), ns not significant, n = 150 boutons per timepoint (d, bouton diameters), ns not significant, n ≥ 9 presynaptic terminals per timepoint (d, single active zone boutons), ns = not significant, ***p ≤ 0.001, n = 150 boutons per timepoint (f, bouton diameters), ns not significant, ***p < 0.001, n ≥ 14 presynaptic terminals per timepoint (f, single active zone boutons). All statistical analysis details with a precise value of ‘n’ are reported in Supplementary Tables 4 and 5 and in Source Data file. Scale bar = 2 μm (b, d, f). Experiments were carried out at 29 °C. Data are represented as mean ± SEM. n = biologically independent samples.