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. 2016 Dec 12;6:38751. doi: 10.1038/srep38751

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Copyright © 2016, The Author(s)

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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HeLa S3/TR cells stably expressing inducible NLS-Lyn (HeLa S3/NLS-Lyn cells) or inducible NLS-Lyn(KD) (HeLa S3/NLS-Lyn(KD) cells) and HeLa S3/TR parental cells were arrested at G1/S phase by 24 h-thymidine treatment, subsequently released into fresh medium for 12 h, and incubated with 1 μg/ml Dox during the last 6 h (see e). (a) Cells were arrested at G1/S phase, released for 6 h, and incubated for 6 h in medium with or without Dox. Whole cell lysates from HeLa S3/NLS-Lyn, HeLa S3/NLS-Lyn(KD), and vector-transfected HeLa S3/TR parental cells were analyzed by Western blotting using anti-Lyn, anti-phosphotyrosine (pTyr), and anti-actin (loading control) antibodies. Full-length blots are presented in Supplementary Fig. S1. (b) Dox-treated HeLa S3/NLS-Lyn cells were stained for DNA. Arrows indicate an anaphase bridge and a lagging chromosome (phenotypes of chromosome missegregation). Scale bar, 10 μm. (c) HeLa S3/NLS-Lyn cells and HeLa S3/NLS-Lyn(KD) cells were treated with Dox for 6 h (see e). The number of cells exhibiting chromosome missegregation was counted. Values are means ± S.D. from more than three independent experiments [HeLaS3/NLS-Lyn (>483 cells), HeLa S3/NLS-Lyn(KD) (>516 cells)], and the significant difference is calculated by the Student’s t-test. P = 0.74 indicates no significant difference. (d) HeLa S3/NLS-Lyn cells were treated with 10 μM PP2 for the last 3 h during Dox treatment (see f). The number of cells exhibiting chromosome missegregation was counted. Values are means ± S.D. from more than three independent experiments [HeLa S3/NLS-Lyn (>426 cells)], and the significant differences are calculated by the Student’s t-test. (e) Schematic depiction of cell synchronization and PP2 treatment. The appearance of mitotic cells was examined after Dox addition by counting the number of H3pS10-positive HeLa S3/NLS-Lyn cells (mitotic index). (f) Schematic depiction of cell synchronization and stepwise PP2 addition. HeLa S3/NLS-Lyn cells were treated with or without 10 μM PP2 for the indicated times. The number of cells exhibiting chromosome missegregation was counted. Significant differences are calculated by the Student’s t-test.