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. 2021 Feb 1;9:634708. doi: 10.3389/fcell.2021.634708

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Copyright © 2021 Dimchev, Lahmann, Koestler, Kage, Dimchev, Steffen, Stradal, Vauti, Arnold and Rottner.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Acute Arp3 removal up-regulates FMNL formin expression enhancing filopodia formation and cell spreading. (A) Representative Western blot of Arp3 as well as FMNL2 and FMNL3 protein levels in Actr3^fl/fl clones with and without tamoxifen treatment, as indicated. GAPDH served as loading control. (B) Quantification of FMNL2 and FMNL3 protein levels from Western blots as shown in (A). Bar charts display arithmetic means of FMNL2 and−3 protein levels normalized to GAPDH, with each FMNL variant (shades of red) being presented as fold-change relative to its respective control (blue). Error bars represent SEMs; data obtained from at least five independently generated cell extracts. Statistics were performed utilizing non-parametric Mann-Whitney rank sum test (***p < 0.001; ** < 0.01). Note the significant elevation of FMNL2 and−3 protein levels upon Arp3 suppression in all three clones. (C) Representative Western blot analysis of FMNL2 and FMNL3 protein amounts in detergent-soluble extracts from DMSO/EtOH- and tamoxifen-treated Actr3^fl/fl cells (clone Arp3.19) after co-transfection with RNAi plasmids individually downregulating FMNL2 and FMNL3 expression or mock-plasmid as control, as indicated. Note the moderate but consistent reduction of FMNL2 and−3 protein levels with and without tamoxifen-induced Arp3 depletion, as indicated (for quantification results see main text). (D) Representative phalloidin stainings of Actr3^fl/fl cells (clone Arp3.19) control or tamoxifen-treated, and combined with mock- or FMNL2/3-RNAi after 15 min of spreading on fibronectin-coated coverslips. (E,F) Quantification of filopodia formation (E) and spreading area (F) of cells as presented in (D); box and whiskers plots as described for Figure 4B. n = total number of cells analyzed from three independent experiments. Statistics were performed utilizing non-parametric, Mann-Whitney rank sum test (***p < 0.001; ** < 0.01; n.s. not significant). Note that Arp3 knockout is accompanied by a pronounced increase in filopodia numbers that are reduced even below Arp3-treatment control levels upon FMNL2/3 RNAi. In addition, the increase in tamoxifen-induced cell area upon 15 min of spreading is also suppressed significantly by FMNL2/3 knockdown.