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. 2021 Feb 4;12:791. doi: 10.1038/s41467-020-20362-2

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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. 2 — a Migration tracks of testis myotubes 33 h APF in 420 min ex vivo culture, treated with DMSO as a control. Different colors represent individual cell tracks. b, c CK666 (100 µM) treatment of a testis 33 h APF in 420 min ex vivo culture. Upon Arp2/3 complex activity inhibition, migration is reduced. mef2-Gal4 drives UAS-LifeAct-EGFP and UAS-mCD8-RFP expression. b Migration tracks of testis myotubes upon CK666 treatment. Different colors represent individual cell tracks. b Life imaging micrographs. mCD8-RFP in green and LifeAct-EGFP in white. The dashed line in c represents the area depicted in 0–420 min. Scale bar, 50 µm. d, e Migration is also mildly reduced by arp3 RNAi. mef2-Gal4 drives UAS-LifeAct-EGFP, UAS-mCD8-RFP, and the RNAi construct UAS-arp3^KK102278 (Vienna v108951). d Migration tracks of testis myotubes upon arp3 RNAi. Different colors represent individual cell tracks. e, e’ Life imaging micrograph. mCD8-RFP (green) is depicted in e. (Note: mononucleated myotubes marked by co-expression of the mCD8-RFP marker are excluded from the nuclei.) Overlay with LifeAct-EGFP (white) in e’. The dashed line in e’ represents the area depicted in 0–350 min. Scale bar, 50 µm. f, g Upon formin suppression through SMIFH2 (10 µM) treatment, migration is completely disrupted. mef2-Gal4 drives UAS-LifeAct-EGFP and UAS-mcd8-RFP expression. f Migration tracks of testis myotubes upon SMIFH2 treatment. Different colors represent individual cell tracks. g, g’ Life imaging micrographs. mCD8-RFP (green) is depicted in g. Overlay with LifeAct-EGFP (white) in g’. The dashed line in g’ represents the area depicted in 0–350 min. Scale bar, 50 µm. h Quantification total migration distance along x-axis. N = 5 testes. Data are presented as mean values ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Statistical testing: one-way ANOVA. i–k Close-ups of front-row myotubes upon different treatments. i Upon DMSO treatment, cell morphology and filopodia composition were not affected. j Arp2/3 suppression by CK666 treatment leads to mild defects. No branched filopodia are built, the overall morphology is unaffected. k Formin suppression by SMIFH treatment leads to strong morphological defects. Cells are contracted, filopodia generate more branches. l CK666 in addition to SMIFH2 co-treatment leads to a loss of branched filopodia. Cells are contracted even stronger.