Fig. 4
From: AURKB as a target in non-small cell lung cancer with acquired resistance to anti-EGFR therapy
Inhibition and silencing of AURKB in cell lines resistant to EGFR TKIs (cont). a Results of flow-cytometry experiments in PC9-ER (p.T790M negative) and PC9-GR4 (p.T790M positive) showing the induction of G2/M arrest and polyploidy after 24 h treatment with barasertib (0.06 µM) or S49076 (0.35 µM). Values shown are means ± SD of three independent determinations (n = 3) and asterisks indicate statistical significance (p < 0.05) in a two-sided Student’s t-test. b Results of flow-cytometry experiments showing the induction of cell death in PC9-ER but not in PC9-GR4 after 24 h treatment with barasertib (0.06 µM) or S49076 (0.35 µM). Values shown are means ± SD of three independent determinations (n = 3) and asterisks indicate statistical significance (p < 0.05) in a two-sided Student’s t-test. c Western blot analysis of poly [ADP-ribose] polymerase 1 (PARP) cleavage in PC9-ER and PC9-GR4 after 24 h with S49076 (0.35 µM). d Acidic beta-galactosidase staining of PC9-ER and PC9-GR4 cells after 48 h with barasertib or S49076, showing the induction of senescence-associated activity in PC9-GR4. Scale bars indicate 50 μm. e Percentage of cells with active acidic beta-galactosidase, as measured by C12FDG fluorescent staining followed by flow cytometry, in PC9-ER and PC9-GR4 cells treated with S49076 for 48 h. f Results of flow-cytometry experiments of PC9-GR1, PC9-GR4-AZD1, H1975 (p.T790M positive), PC9-GR3, and PC9-GR1-AZD3 (p.T790M negative), showing the induction of apoptosis only in p.T790M-negative cells after 24 h treatment with barasertib (0.06 µM). Values shown are means ± SD of three independent determinations (n = 3) and asterisks indicate statistical significance (p < 0.05) in a two-sided Student’s t-test. g Acidic beta-galactosidase staining of the same cells after 48 h with barasertib, showing the induction of senescence-associated activity exclusively in p.T790M-positive cells. Scale bars indicate 10 μm. h Changes in the mRNA levels of selected genes after a 6-day treatment with S49076 or barasertib. Values shown are means ± SD of three independent determinations (n = 3) and asterisks indicate statistical significance (p < 0.05) in a two-sided Student’s t-test. i Proposed model to explain the effects of S49076 and barasertib in EGFR-TKI-resistant cells
