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. 2015 Feb 12;6(2):e1637. doi: 10.1038/cddis.2015.2

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Copyright © 2015 Macmillan Publishers Limited

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International Licence. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons licence, users will need to obtain permission from the licence holder to reproduce the material. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0

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PAK1 kinase activity inhibition enhances sunitinib sensitivity in RCC cells. (a) Western blot analysis of PAK1, p-PAK1(T423) and GAPDH for ACHN cells stably transfected with NS-shRNA and PAK1-shRNA-1 without or with sunitinib (5 μM) treatment for 48 h (left panel), for ACHN cells stably transfected with control (empty vector) or PAK1-K299R without or with sunitinib (5 μM) treatment for 48 h (middle panel), for OS-RC-2 cells in the presence with IPA3 (7.5 μM) with or without sunitinib (5 μM) treatment for 48 h (right panel). GAPDH was used as a loading control. The numbers shown are the ratios of the intensities of the bands for p-PAK1(T423) divided by the intensities of the bands for GAPDH, normalized to 1.00 for ACHN cells transfected with NS-shRNA, and OS-RC-2 cells without treatment with sunitinib, respectively. (b) Analysis of self-renewal of ACHN and OS-RC-2 cells with above-mentioned treatment. Data are represented as means±S.D. of triplicate experiments. *P<0.05 versus sunitinib. (c) Flow cytometry analysis of ALDH1, CD73 and CD146 for ACHN and OS-RC-2 cells with above-mentioned treatment from generations G3 spheres. Data are represented as means±S.D. of triplicate experiments. *P<0.05 versus sunitinib. (d–f) Cell proliferation assay (d), annexin V/PI staining assay (e) and caspase 3/7 activity (f) for ACHN and OS-RC-2 cells with above-mentioned treatment. Data are represented as means±S.D. of triplicate experiments. *P<0.05

PAK1 kinase activity inhibition enhances sunitinib sensitivity in RCC cells. (a) Western blot analysis of PAK1, p-PAK1(T423) and GAPDH for ACHN cells stably transfected with NS-shRNA and PAK1-shRNA-1 without or with sunitinib (5 μM) treatment for 48 h (left panel), for ACHN cells stably transfected with control (empty vector) or PAK1-K299R without or with sunitinib (5 μM) treatment for 48 h (middle panel), for OS-RC-2 cells in the presence with IPA3 (7.5 μM) with or without sunitinib (5 μM) treatment for 48 h (right panel). GAPDH was used as a loading control. The numbers shown are the ratios of the intensities of the bands for p-PAK1(T423) divided by the intensities of the bands for GAPDH, normalized to 1.00 for ACHN cells transfected with NS-shRNA, and OS-RC-2 cells without treatment with sunitinib, respectively. (b) Analysis of self-renewal of ACHN and OS-RC-2 cells with above-mentioned treatment. Data are represented as means±S.D. of triplicate experiments. *P<0.05 versus sunitinib. (c) Flow cytometry analysis of ALDH1, CD73 and CD146 for ACHN and OS-RC-2 cells with above-mentioned treatment from generations G3 spheres. Data are represented as means±S.D. of triplicate experiments. *P<0.05 versus sunitinib. (d–f) Cell proliferation assay (d), annexin V/PI staining assay (e) and caspase 3/7 activity (f) for ACHN and OS-RC-2 cells with above-mentioned treatment. Data are represented as means±S.D. of triplicate experiments. *P<0.05