KCNF1 promotes lung cancer by modulating ITGB4 expression

Figures

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  Fig. 1. Expression of KCNF1 is elevated and associated with poor outcomes in NSCLC. A Analysis of TCGA dataset showed increased KCNF1expression in lung adenocarcinoma. B Kaplan–Meier survival analysis of TCGA lung adenocarcinoma dataset showed that increased expression of KCNF1 is correlated with poor survival. C, D Elevated KCNF1 expression is associated with reduced overall survival using univariate analysis and multivariate analysis after adjusting confounding factors including gender, AJCC stage N, and smoking using Kaplan–Meier Plotter. E Expression of KCNF1 in fresh frozen human lung cancer tissues and adjacent uninvolved lung tissues was analyzed by immunoblotting using anti-KCNF1 and anti-GAPDH. U uninvolved, T tumor. F Quantification of KCNF1 expression analyzed in (E).

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  Fig. 2. Silencing of KCNF1 reduces lung cancer tumorigenesis. A Lysates of Beas2B, A549, H23, and H2122 cells were analyzed by immunoblotting using anti-KCNF1 and anti-GAPDH antibodies. B Lysates of A549 and H23 cells transduced with lentiviral vectors expressing a control shRNA or two different shRNAs against KCNF1 were analyzed by anti-KCNF1 and anti-GAPDH. C, D Proliferation of transduced cells was analyzed. E, F Migration of transduced cells was analyzed. G, H Transduced cells were injected into the flanks of nude mice. Tumor volume was monitored. All data are mean ± SD. *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.00005.

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  Fig. 3. Ectopic expression of KCNF1 promotes cell proliferation. A Lysates of Beas2B and HSAEC cells transfected with a control vector or a vector expressing FLAG and HA double tags at the N-terminus (FH-KCNF1) were analyzed by anti-HA and anti-Actin. B, C Proliferation of transfected cells was analyzed. All data are mean ± SD. **P < 0.005; ***P < 0.0005; ****P < 0.00005.

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  Fig. 4. Silencing of KCNF1 re-establishes basement membrane integrity. A Bright field images of the spheroids from control A549 cells and A549 cells with knockdown of KCNF1. B Spheroids of control A549 cells and A549 cells with knockdown of KCNF1 were analyzed by IF using anti-KCNF1 and anti-laminin V antibodies. Cells were also stained with DAPI; scale bar, 20 μm at 40X objective. C Spheroids of control A549 cells and A549 cells with knockdown of KCNF1 were analyzed by IF using anti-phosphorylated ERM and anti-laminin V antibodies. Cells were also stained with DAPI; scale bar, 50 μm at 20X objective.

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  Fig. 5. KCNF1 is primarily localized in the nucleoplasm of NSCLC cells. A A549 cells were stained with anti-KCNF1 in the presence of GST or GST-KCNF1 protein and DAPI, and analyzed by a confocal fluorescence microscope. B Beas2B, H23, and H1299 cells were stained with anti-KCNF1 and DAPI, and analyzed by a confocal fluorescence microscope. A, B scale bar, 5 μm at 63X objective.

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  Fig. 6. Silencing of KCNF1 increases cisplatin sensitivity. A NSCLC cell lines, A549, H661, H1299, H23, H2122, and H1975, were treated with DMSO or 10 μM cisplatin for 72 hours. Cell growth was analyzed by SRB staining. The cell growth with DMSO for each cell line was set at 100. B KCNF1 expression in NSCLC cell lines was analyzed by immunoblotting. C Lysates of H1299, H661, and H1975 transfected with a control siRNA or an siRNA targeting KCNF1 were analyzed by immunoblotting using anti-KCNF1 and anti-GAPDH. D–F H1299, H661, and H1975 cells were transfected with a control siRNA or an siRNA targeting KCNF1. Transfected cells were treated with different concentrations of cisplatin (0.01–50 μM) for 72 hours. Cell growth was analyzed by SRB staining. IC50 of each cell line transfected with siControl or siKCNF1 was calculated.

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  Fig. 7. KCNF1 positively regulates ITGB4 expression and downstream signaling. A Lysates of A549 and H23 cells transduced with lentiviral vectors expressing a control shRNA or an shRNA against KCNF1 were analyzed by anti-KCNF1, anti-ITGB4, anti-p-FAK, anti-FAK, anti-p-AKT, anti-AKT, or anti-Actin. B Lysates of A549 and Beas2B cells with forced expression of FLAG and HA-tagged KCNF1 (FH-KCNF1) were analyzed by immunoblotting using anti-HA, anti-ITGB4, anti-p-FAK, anti-FAK, anti-p-Akt, anti-Akt, or anti-Actin. C A549 cells transduced with shControl or shKCNF1were transfected with a control vector or a vector expressing Myc-His-ITGB4. Levels of ITGB4, Myc-His-ITGB4, p-FAK, FAK, p-Akt, Akt, KCNF1, Actin, and GAPDH were analyzed by immunoblotting.