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. 2019 Nov 29;9:17889. doi: 10.1038/s41598-019-54478-3

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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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Knockdown of NCAPH induces S and G_2/M phase arrest and apoptosis. (A) MIA PaCa-2 cells were transfected with control siRNA and NCAPH siRNA and then subjected to flow cytometric analysis at 24, 48, 72 and 96 h. The flow cytometry plots and data are representative of at least three separate experiments. (B) Bar graph showing the percentages of cells in sub-G₁ (apoptosis, red), G₁ (blue), S (orange), and G₂/M (green) phases. The experiments were repeated three times, and mean values of the results are presented. ***P < 0.0001, two-way analysis of variance. (C) Western blot analysis was used to determine the levels of NCAPH, cyclin A, phospho-histone H3, caspase-3, PARP, and β-actin in MIA PaCa-2 cells after transfection with control siRNA or NCAPH siRNA for 24, 48, and 72 h. Cell lysates were immunoblotted with the indicated antibodies. (D) Annexin V staining was performed to measure apoptosis using flow cytometry. Z-VAD-Fmk (20 μM) was treated with cells for 24 h. LL (lower left) represents live, LR (lower right) represents early-apoptotic cells, UR (upper right) represents late stage apoptotic cells. (E) Western blot analysis was used to determine the levels of NCAPH, caspase-3, PARP, and β-actin in MIA PaCa-2 cells after transfection with control siRNA or NCAPH siRNA. Cells were treated with Z-VAD-Fmk (20 μM) for 24 h.