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. 2019 May 16;10:2193. doi: 10.1038/s41467-019-10229-6

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© The Author(s) 2019

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. 4 — WASH (Wiskott–Aldrich syndrome protein and SCAR homologue) inactivation suppresses the Ptp4EPt10D tube shape phenotype. a Confocal frames showing the dorsal trunk (DT) of living wild-type and wash¹⁸⁵ mutant embryos expressing btl>ANF-GFP (green). wash¹⁸⁵ mutant embryos retain luminal ANF-GFP (lowest row) or delay clearance (middle row). Time points represent hours after egg laying (AEL). b Plots showing the percentage of wild-type (n = 48) and wash¹⁸⁵ (n = 57) mutant embryos that complete ANF-GFP luminal clearance (20 h AEL). *P < 0.005 (unpaired two tailed t tests). c Plots depicting the average time (hours) of luminal ANF-GFP clearance completion in wild-type (n = 19) and wash¹⁸⁵ (n = 19) mutant embryos. Error bars show s.e.m. **P < 0.001 (unpaired two tailed t test). d, e Plots depicting the percentage of wild-type (n = 27), wash¹⁸⁵ (n = 32) and wash¹⁸⁵;btl>Wash (n = 52) embryos completing the ANF-GFP luminal clearance (d) or gas filling (e). Re-expression of Wash specifically in the tracheal cells of wash¹⁸⁵ mutants rescues the ANF-GFP luminal clearance and gas-filling defects. *P < 0.02; **P < 0.009 (unpaired two tailed t tests). f Confocal image depicting the DT of an embryo expressing endogenous YFP-tagged Rab7, stained with anti-WASH (magenta), anti-GFP (green) and DAPI (blue). Inset denotes zoomed view of a late endosome (arrowhead). Arrowheads indicate colocalization of WASH and Rab7. g, h Confocal images showing the tracheal DT of stage 17 wild-type, wash¹⁸⁵, Ptp4EPtp10D and Ptp4EPtp10D;wash¹⁸⁵ mutant embryos stained for Verm (green) and the AJ marker (DECad, magenta). Single sections (upper row) (g) and Z-stack maximum projections (lower row) (h). i Plots depicting the average DT diameter of wild-type (n = 12), wash¹⁸⁵ (n = 14), Ptp4EPtp10D (n = 17) and Ptp4EPtp10D;wash¹⁸⁵ embryos (n = 21). The DT diameter phenotype of Ptp4EPtp10D is significantly restored in the Ptp4EPtp10D;wash¹⁸⁵ triple mutants. *P < 0.018 between Ptp4EPtp10D and Ptp4EPtp10D;wash¹⁸⁵ embryos (unpaired two tailed t test). The boxplots show the median (internal line) and the range of the data is from 25th to 75th percentile. The bars present maxima and minima values. Scale bars, 10 μm