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. 2020 Aug 20;11(8):670. doi: 10.1038/s41419-020-02908-w

Fig. 4. NANOG mediates the regulation of IRX4 on cancer stem-like properties and gefitinib resistance.

Fig. 4

a PC-9/GR cells were exposed to BBI503 (0.1 and 1μmol/L) for 48 h and then harvested for western blotting to detect the NANOG and CD133 expression. b Treatment with BBI503 at indicated concentrations for 48 hours suppressed the growth of PC-9/GR cells in a dose-dependent manner. ***P < 0.001, ****P < 0.0001. c Effect of combined treatment with BBI503 (0.1 μM) and gefitinib on growth of PC-9/GR cells was evaluated, and the IC50 against gefitinib was calculated (mean ± SD; n = 3; ***P < 0.001). d PC-9/GR cells were transfected with NANOG siRNA (siNANOG) or control siRNA (siCtrl). Total cell lysate proteins were extracted for western blot analysis of NANOG and CD133 expression using specific antibodies. e–f The detection of clonal formation and cell viability after silencing NANOG expression (mean ± SD; n = 3; ***P < 0.001). g PC-9/GR cells were transfected with siNANOG or siCtrl, after 6 h, the cells were exposed to various concentrations of gefitinib (0, 6.25, 12.5, 25, 50 μM) for another 48 h. Cell proliferation was determined by MTT assay, and the IC50 against gefitinib was calculated. Data represent the mean ± SD of three replicate determinations, ***P < 0.001. h PC-9/GR cells were infected with lentivirus carrying shIRX4 or shNC for 24 h, and then NANOG was overexpressed by transfecting with the pcDNA3.1-NANOG plasmid or the empty vector (pcDNA3.1) for another 48 h, and the expressions of IRX4, NANOG and CD133 were evaluated by western blotting. i PC-9/GR cells were infected with lentivirus carrying shIRX4 or shNC for 24 h, and then transfected with pcDNA3.1-NANOG plasmid or the empty vector, at 6 h post-transfection, the cells were treated with various concentrations of gefitinib (0, 3.75, 7.5, 15, 30, 60 μM) for another 48 h. Cell proliferation was determined by CCK8 assay, and the IC50 against gefitinib was calculated. Data represent the mean ± SD of three replicate determinations, ***P < 0.001, ****P < 0.0001.