Fig. 4

EGFR and PTK2 co-inhibition induce apoptosis in PC-9/PEM clone1. a Representative immunoblots of phosphorylated and total PTK2 in PC-9 and PC-9/PEM clone1 cells and the ratio of phosphorylated PTK2 to β-actin. Data are means (SD), n = 3. *, P < 0.05 by Student’s t-test. b Viabilities of PC-9, PC-9/PEM, and PC-9/PEM clone1 cells treated with erlotinib or osimertinib at the indicated concentrations for 96 h. Data are means (SD), n = 3. PC-9 vs. PC-9/PEM, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, PC-9 vs. PC-9/PEM clone1, ^#, P < 0.05; ^##, P < 0.01; ^###, P < 0.001; ^####, P < 0.0001 by one-way ANOVA and Tukey’s HSD multiple comparisons test. c Viability of PC-9/PEM clone1 cells with or without defactinib and erlotinib (upper) or osimertinib (lower) at the indicated concentrations for 96 h. Data are means (SD), n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data for cells with and without defactinib were compared by Student’s t-test. d Viability of PC-9/PEM clone1 cells with or without 1 μM defactinib and EGFR-TKI (0.01 μM gefitinib, 0.03 μM erlotinib, 0.001 μM afatinib, or 0.01 μM osimertinib). Data are means (SD), n = 3. **, P < 0.01; ****, P < 0.0001, EGFR-TKI + Def vs. Def; ^###, P < 0.001; ^####, P < 0.0001, NS, not significant by one-way ANOVA and Tukey’s HSD multiple comparisons test. e Immunoblots of the phosphorylated and total levels of the indicated proteins in PC-9 and PC-9/PEM clone1 cells treated with 3 μM defactinib or DMSO for 96 h. f Representative immunoblots of the indicated proteins in PC-9 and PC-9/PEM clone1 cells with or without 1 μM defactinib (Def) and 0.1 μM osimertinib (Osi). Ratios of phospho-Akt to β-actin (upper) and cleaved PARP to β-actin (lower). Data are means (SD), n = 3. **, P < 0.01, ***, P < 0.001, NS, not significant by one-way ANOVA and Tukey’s HSD multiple comparisons test. g Immunoblots of phosphorylated and total PTK2 in PC-9/PEM clone1 cells treated with or without 1 μM defactinib, 1 μM PD173074 or 1 μM BLU-554 for 72 h. h Immunoblots of phosphorylated and total PTK2 in PC-9/PEM clone1 cells transfected with siRNAs against PTK2. The siCtrl was used as a negative control. i Viability of PC-9/PEM clone1 cells transfected with the indicated siRNAs and treated with osimertinib at the indicated concentrations for 96 h. Data are means (SD), n = 3. (siCtrl vs. si#1, *, P < 0.05; **, P < 0.01), (siCtrl vs. si#2, ^#, P < 0.05; ^##, P < 0.01) by one-way ANOVA and Tukey’s HSD multiple comparisons test