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. 2019 Sep 23;9:13703. doi: 10.1038/s41598-019-50275-0

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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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Depletion of CdK5 abrogates the effects of EGF on ADD1 T724 phosphorylation, lamellipodia formation, and cell migration. (a) MDA-MB-231 cells were serum-starved for 16 h and treated with (+) or without (−) 40 ng/ml EGF for 15 min. An equal amount of the whole cell lysates was analyzed by immunoblotting with antibodies as indicated. (b) MDA-MB-231 cells were infected with lentiviruses expressing shRNAs to Cdk5 (shCdk5 clone#1 and #2) or luciferase (shLuc) as a control. The cells were serum-starved for 16 h and treated with (+) or without (−) 40 ng/ml EGF for 15 min. (c) MDA-MB-231 cells described in (b) were plated on collagen-coated coverslips, and treated with (+) or without (−) 200 ng/ml EGF for 6 h. The percentage of cells with lamellipodia in the total number of counted cells was measured (n ≥ 200). Values (means ± s.d.) are from three independent experiments. **P < 0.01. (d) MDA-MB-231 cells described in (b) were suspended in serum-free medium and subjected to the cell migration assay with (+) or without (−) 40 ng/ml EGF in the lower chambers. After 6 h, the migrated cells were fixed, stained, and counted using a light microscope. Representative micrographs are shown. Bars, 100 μm. The number of migrated cells were measured and expressed as percentage relative to the control in the absence of EGF. Values (means ± s.d.) are from three independent experiments. **P < 0.01.