A tight balance of Karyopherin β1 expression is required in cervical cancer cells

Figures

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  Fig. 1. Kpnβ1 localizes to the nucleus and nuclear membrane in cervical cancer cells and enhances the nuclear import of known Kpnβ1 cargoes. a Immunofluorescence analysis was used to determine GFP expression across the HeLa and CaSki EGFP and Kpnβ1-EGFP stable cell lines. Representative GFP-Alexa 488 images are shown for each cell line. Enlarged images in the top right-hand corner show perinuclear localization in representative Kpnβ1-EGFP cells. b Western blot analysis was used to determine endogenous Kpnβ1 (97 kDa) and exogenous Kpnβ1-EGFP (129.7 kDa) expression levels across the HeLa and CaSki EGFP and Kpnβ1-EGFP cell lines. The first lane represents protein harvested from untransfected cells. GAPDH was used as a control for loading. c - f Stable expression of Kpnβ1-EGFP in HeLa cells resulted in a significant increase in NFAT (c), AP-1 (d), PMA-stimulated NFκB p65 (e) and p53 (f) promoter-driven luciferase activity. Results shown represent the mean ± SEM (*p < 0.05)

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  Fig. 2. Overexpression of Kpnβ1 results in reduced proliferation of cervical cancer and normal epithelial cells. a, b Trypan Blue assays revealed a significant reduction in the number of live HeLa (a) and CaSki (b) cells expressing Kpnβ1-EGFP compared to EGFP. Cells were counted for a period of up to 4 days post plating. c, d MTT cell proliferation assays reveal a significant reduction in the anchorage-independent proliferation of HeLa (c) and CaSki (d) cells stably overexpressing Kpnβ1. Cells growing adherently were plated in 1% methylcellulose-containing media into 96-well plates coated in 12 mg/ml poly-HEMA. Cell proliferation was assayed by the addition of the MTT reagent for a period of up to 8 days. Results shown represent the mean ± SEM (*p < 0.05). e Western blot analysis reveals moderate Kpnβ1 overexpression in ARPE-19 and hTERT-RPE-1 normal epithelial cell lines. f Trypan blue assays show a significant reduction in ARPT-19 and hTERT-RPE-1 cell number 96 h after transient Kpnβ1 overexpression. Results shown represent the mean ± SEM (*p < 0.05)

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  Fig. 3. Overexpression of Kpnβ1 results in a delay in cell cycle progression. a Protein was harvested from HeLa and CaSki EGFP and Kpnβ1-EGFP cells and cell cycle markers investigated by western blot analysis. Cyclin A, cyclin D1 and pHistoneH3 were all expressed at slightly higher levels in the overexpressing cells compared to the EGFP control cells. GAPDH was used to control for protein loading. b Flow cytometric analysis of HeLa EGFP and HeLa Kpnβ1-EGFP cells following synchronization into late G1 and release into S phase. Cells were synchronized with 2 mM thymidine, harvested at various time points post release and examined by FACS analysis. An asynchronous culture of cells (async) was used for comparison. c and d Line graphs represent quantification of the percentage of cells in each phase of the cell cycle for EGFP (b) and Kpnβ1-EGFP (c) cells. Arrows represent the approximate point where 50% cells had exited the G1 phase. Results shown represent the mean ± SD. e Western blot analysis was used to determine the expression levels of cell cycle associated proteins at various time points post release following thymidine synchronization, in HeLa EGFP and Kpnβ1-EGFP cells. Protein…

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  Fig. 4. Overexpression of Kpnβ1 results in changes in the morphology and adhesion properties of cervical cancer cells. a Phase contrast images showing HeLa EGFP and Kpnβ1-EGFP cells, taken 48 h post plating. Cells were viewed at 20 x magnification using the Zeiss Primovert inverted phase microscope. b Quantification of relative HeLa cell area ± SEM of forty cells from each condition was performed using the AxioVision 4.7 software (*p < 0.05). c Fluorescent staining of polymeric F-actin using phalloidin (red) in EGFP and Kpnβ1-EGFP expressing HeLa cells. DAPI stain was used to visualize the cell nuclei (blue). d Quantification of the number of cytoplasmic protrusions from the captured fluorescent images. Results shown represent the mean ± SEM over fifteen fields of view (*p < 0.05). e Relative cell adhesion of HeLa EGFP and Kpnβ1-EGFP cells. Adherent cells (on uncoated tissue culture plates) were fixed (after removing non-adherent cells by washing) and stained with 0.5% crystal violet solution. Cells over ten fields of view, viewed at 10 x magnification, were counted using ImageJ and normalized to unwashed cells. Results shown represent the mean ± SEM (*p < 0.05). f Western blot analysis…

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  Fig. 5. Cisplatin sensitivity in Kpnβ1 overexpressing cells. A The IC50 value for cisplatin was determined for HeLa EGFP and HeLa Kpnβ1-EGFP cells. B Cell viability for EGFP and Kpnβ1-EGFP cells was analyzed 48 h after cisplatin treatment via an MTT assay, and results were normalized to the viability of the untreated cells. Results shown for A and B represent the mean ± SEM (*p < 0.05). C PARP cleavage was analyzed as an indication of apoptosis in both EGFP and Kpnβ1-EGFP cells 48 h after cisplatin treatment. GAPDH was used as a control for loading. D Densitometric quantification of C-PARP relative to GAPDH from two independent experiments. E HeLa EGFP and Kpnβ1-EGFP cells were treated with cisplatin for 24 h, followed by western blot analysis to determine the levels of γH2AX, p53, p21 and Mcl-1. GAPDH was used as a control for loading. F Nuclear and cytoplasmic proteins were isolated from HeLa EGFP and Kpnβ1-EGFP cells and p53 and p21 levels determined by western blot analysis. TBP and GAPDH were used to control for even nuclear and cytoplasmic protein loading, respectively. G HeLa EGFP (a) and Kpnβ1-EGFP (b) cells were co-treated with Cisplatin and the p53 inhibitor Pifithrin α, and…