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. 2018 Sep 6;9:3613. doi: 10.1038/s41467-018-05808-y

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© The Author(s) 2018

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. 6 — Phosphorylation by Cdk5 drives the degradation of lamin A/C after Nestin knockdown. a, b FRAP anaysis of GFP-lamin A/C in control and Nestin-knockdown A549 cells. a Representative fluorescence images showed (from left to right): pre-bleach image; image taken immediately after bleaching; and two frames (15 and 30 min) from a time-lapse image series taken at 20-s intervals over half an hour. Scale bars, 5 μm. b The mean normalized FRAP data measured from the bleached regions (n = 5). c The average mobile fractions of the lamin A/C proteins were calculated from the FRAP data shown in b (n = 5). d, e Immunostaining and immunoblotting analysis of lamin A/C status. A549 cells of control or Nestin-knockdown were treated with BMS-265246 (1 μM), roscovitine (20 μM), or MK-2206 (3 μM). After 24 h, cells were stained with anti-lamin A/C antibody (red) and DAPI (blue) (Scale bars, 5 μm) (d) or immunoblotted with the indicated antibodies (e). f The effects of Nestin knockdown on Cdk5. Cdk5 was immunoprecipitated in control or Nestin-knockdown A549 and H1299 cells, and kinase assays were conducted using a Cdk5 substrate peptide, as described in the Methods section. g Representative immunofluorescence data for pS22 lamin A/C or pS392 lamin A/C (green) plus Nestin (white) in control and Nestin-knockdown A549 cells. Scale bars, 5 μm. h Immunoblotting analysis of lamin A/C phosphorylation levels affected by Nestin and Cdk5 knockdown. A549 cells were transfected with the indicated shRNAs, and proteins were extracted and subjected to immunoblotting. i Confocal microscopy was employed to determine the subcellular localization of WT GFP-lamin A/C, compared with GFP-lamin A/C-S392D. A549 cells transfected with the indicated constructs were treated with 25 ng/ml leptomycin B (LMB) for 6 h, and then harvested, fixed, and stained with DAPI (blue). Scale bars, 10 μm. j Illustration of the Nestin–Cdk5–lamin A/C axis in the regulation of tumor senescence. The quantified results were presented as mean ± SEM of five independent experiments (b, c), as assessed using unpaired t-test (c). **P < 0.01