Fig 1: NANOG mediates the regulation of IRX4 on cancer stem-like properties and gefitinib resistance.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.
Fig 2: Downregulation of IRX4 increases gefitinib cytotoxicity and inhibits expression of NANOG and CD133 in a PC-9/GR xenograft mouse model.a Flow charts of the establishment of xenograft mouse model and gefitinib was administered during day 7 – day 21. b–c Tumor sizes and weight were presented as mean ± SD; n = 5; ns, not significant, *P < 0.05, **P < 0.01, ****P < 0.0001. d Macroscopic appearance of PC- 9/GR xenografts of each group. e Whole protein cell lysates were prepared randomly from 3 tumors per group for western blotting to detect the indicated proteins (mean ± SD; *P < 0.05, ***P < 0.001, ****P < 0.0001).
Fig 3: Inhibition of IRX4 decreases NANOG levels and stemness of NSCLC cells.a PC-9/GR cells were infected with lentivirus for 72 h. Note: Numbers represent MOI values; lentivirus concentration: 4.0 × 108TU/mL. b PC-9/GR cells were infected with lentivirus for 48 h, then the total RNA was extracted, and mRNA level of IRX4 was detected by real-time PCR (mean ± SD; n = 6; ***P < 0.001). c A total of 5 × 103 cells were seeded into 6-well ultra low-attachment plates (Corning, NY, USA) and incubated in DMEM/F12 (Biological Industries) supplemented with EGF(20 ng/mL), FGF-basic(20 ng/mL) and B27 (20 µL/mL) for two weeks. Cell spheres characterized by tight, spherical, non-adherent colonies of more than 90µm in diameter were observed and counted (Scale bar: 100 µm; original magnification: ×100; representative images from three experiments; mean ± SD; **P < 0.01). d The PC-9/GR cells were transfected with siIRX4 or siCtrl, after 48 h the percentage of CD133+ cells were analyzed by flow cytometry (mean ± SD; n = 3; ***P < 0.001). e PC-9/GR cells were transfected with control small interference RNA (siCtrl) or IRX4-specific siRNA (siIRX4). After 48 h, the relative mRNA levels of IRX4, NANOG, Notch1 and CTNNB1 were measured by QRT-PCR (mean ± SD; n = 6; **P < 0.01, ***P < 0.001). f PC-9/GR cells were transfected with siCtrl or siIRX4. After 48 h, western blotting was performed to detect the expression of IRX4, CD133, NONOG, Notch1, and ß-catenin, and the relative intensity of these bands was analyzed (mean ± SD; n = 3; *P < 0.05, ***P < 0.001). The data were representatives of 3 independent experiments. g–h PC-9/GR cells were infected with lentivirus for 72 h, the expression of IRX4, NANOG, Sox2, Oct4, ABCG2 and ALDH1A1 was detected by western blotting, and the relative intensity of each protein was calculated and analyzed by ChemiScope analysis software (mean ± SD; n = 3; **P < 0.01, ***P < 0.001, ****P < 0.0001). i PC-9 cells were transfected with empty pcDNA3.1 vectors, or pcDNA3.1-IRX4 for 48 h. The protein levels of IRX4, CD133, MDR1 and NANOG were determined by western blotting. j NANOG transcriptional activity was measured after PC-9/GR cells were transfected with siIRX4 or siCtrl for 48 h. Results are shown as histogram showing quantitative values from triplicate experiments (mean ± SD; n = 3; *P < 0.05). k After transfetcted with siCtrl or siIRX4 for 6 h, PC-9/GR cells were treated with gefitinib (0.1, 1, 10µmol/L) for 48 h, and then western blot analysis was performed to assess the expression of IRX4 and NANOG.
Fig 4: IRX4 expression is upregulated by gefitinib exposure.a IRX4 protein level in LUAD cells was detected by western blotting. The IRX4 expression level relative to GAPDH was quantified with a ChemiScope analysis (mean ± SD; n = 3; ****P < 0.0001). b–c PC-9 and PC-9/GR cells were respectively stimulated with various concentrations of gefitinib for 48 h. MTT assay were carried out to determine the IC50 value against gefitinib. d PC-9 and PC-9/GR cells were treated with various concentrations of gefitinib for 10 days. The colony formation was detected by crystal violet staining. e HE staining was performed to detect the cell morphology of PC-9 and PC-9/GR cells (Scale bar: 100 µm; original magnification: ×100; representative images from three experiments). f The mRNA levels of IRX3, IRX4 and IRX5 were evaluated by QRT-PCR (mean ± SD; n = 6; ***P < 0.001). g IRX4 protein levels in PC-9 and PC-9/GR cells were assessed by western blotting. The relative intensity of IRX4 was evaluated by ChemiScope analysis software (mean ± SD; n = 3; **P < 0.01). h Nuclear and cytoplasmic fractions of PC-9 and PC-9/GR cells were prepared, and IRX4 expression was detected by western blotting. GAPDH and Histone-H3 were used respectively as cytoplasm and nucleus loading control. i The steps of inducing gefitinib-resistant PC-9 cells: PC-9 cells were first treated with gefitinib at the concentration of 20 nM for 1 week. A small amount of remaining cells were then treated for another 2 days with a concentration of 50 nM which was sufficient to kill nearly all parental cells. The remaining few cells were cultured continuously in the absence of gefitinib for 2 weeks. Then the cells were sequentially treated with gefitinib at the concentration of 500 nM for 1 week, 1 µM for another 1 week and 10 µM for the last 1 week. j The PC-9 cells morphology of different time points stimulated with gefitinib were determined by HE staining (Scale bar: 100 µm; original magnification: ×100; representative images from three experiments). k IRX4 protein levels at day 8, 23, 28, 33 and 40 were determined by western blotting. l MTT assay was carried out to determine IC50 value against gefitinib of PC-9-GR cell. m Western blot analysis of IRX4 protein levels in PC-9 and PC-9-GR cells. The relative intensity of IRX4 was evaluated by ChemiScope analysis software (mean ± SD; n = 3; ***P < 0.001).
Fig 5: Combined treatment with 1,25D and gefitinib exhibits increased proliferation suppression of PC-9/GR cells.a Cells were exposed to various concentrations of gefitinib and gefitinib + 1,25D (100 nM) for 2 days. Cell proliferation was determined by the MTT assay. IC50 value against gefitinib was analyzed. Data represent the mean ± SD of three replicate determinations. ****P < 0.0001. b PC-9/GR cells were seeded into 6-well plates (n = 3 per group). The next day, the cells were treated with vehicle (control), 1,25D alone (50 nM), gefitinib alone (1 µM and 5 µM), or the combination. Treatments were repeated every 3 days. Colony formation was assessed by crystal violet staining. The colony numbers were assessed with ImageJ software, and the clonal formation efficiency was calculated (mean ± SD; n = 3; *P < 0.05, ***P < 0.001). c PC-9/GR cells were exposed to vehicle, 1,25D (100 nM), gefitinib (2 µM) or 1,25D + gefitinib for 48 h and stained for Edu (Scale bar: 100 µm; original magnification: ×100; representative images from three experiments). The cell proliferation rate was calculated as a percentage of Edu-positive nuclei to total nuclei (mean ± SD; n = 3; **P < 0.01, ***P < 0.001). d PC-9/GR cells were exposed to gefitinib (0.1 µM and 1 µM) or 1,25D + gefitinib for 48 h and western blotting was performed to detect the expression of IRX4 and NANOG.
Supplier Page from Abcam for Anti-IRX4 antibody