Fig 2: NA49 sensitized tumors in an NCI-H460 and NCI-H1650 cells-injected xenograft mouse model with cisplatin and ge-fitinib. (A) NCI-H460 and NCI-H1650 cells were injected subcutaneously into BALB/c nude mice (n = 6/group). Xenografted mice were treated 7 times with NA49 (20 mg/kg) intraperitoneal treatment in combination with four injections of cisplatin (2 mg/kg) or gefitinib (5 mg/kg). (B) Tumor size was measured every other day. Results are the mean and standard error. (C) Ki67 staining (D) and immunohistochemistry of HSP27 were performed using tumor tissues. Results are the mean and standard error (*p < 0.05, *** p < 0.001 vs. untreated-control; # p < 0.05, ## p < 0.01, ### p < 0.001 ### vs. cisplatin or gefitinib alone).

Fig 3: HSP27 cross-linking activities of NA49 and effect of gefitinib in EGFR mutant-type lung cancer cell lines. (A) NCI-H1650 and NCI-H1975 cells were treated with NA49 (0, 5, 10, and 20 μM) or gefitinib (0, 10, 20, and 30 μM) for 24 h, and cell lysates were detected by Western blot analysis. Protein levels were quantified using Image J software. The data were expressed as the fold change relative to the control and normalized to β-actin in graph. (B) NCI-H1650 and NCI-H1975 cells were treated with NA49 (0, 5, 10, and 20 μM) or gefitinib (0, 10, 20, and 30 μM) for 48 h, and cell death was analyzed by flow cytometry after propidium iodide (PI) staining. Results are the mean and standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated control cells).

Fig 4: HSP27 cross-linking activities of NA49 and effect of cisplatin or gefitinib in EGFR wild-type lung cancer cell lines. (A) NCI-H460 and A549 cells were treated with NA49 (0, 5, 10, and 20 μM), cisplatin (0, 3, 5, and 10 μM), or gefitinib (0, 10, 20, and 30 μM) for 24 h, and cell lysates were detected by Western blot analysis. Protein levels were quantified using Image J software. The data were expressed as the fold change relative to the control and normalized to β-actin in graph. (B) NCI-H460 and A549 cells were treated with NA49 (0, 5, 10, and 20 μM), cisplatin (0, 3, 5, and 10 μM), or gefitinib (0, 10, 20, and 30 μM) for 48 h, and cell death was analyzed by flow cytometry after treatment with propidium iodide (PI). Results are the mean and standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated control cells.).

Fig 5: J2 and NA49 showed sensitization effects on EGFR mutant-type lung cancer cells in combination with gefitinib. (A) HCC827 and PC9 cells were treated with J2 (10 μM) and NA49 (10 μM) for 24 h, with or without gefitinib (10 nM), and cell lysates were detected by Western blot analysis. Protein levels were quantified using Image J software. The data were expressed as the fold change relative to the control and normalized to β-actin in the graph. (B) Cell death was analyzed by flow cytometry after propidium iodide (PI) staining. Results are the mean and standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p< 0.001 vs. untreated-control; # p < 0.05, ## p < 0.01, ### p< 0.001 vs. gefitinib alone). (C) NCI-H1650 cells were treated with J2 (10 μM) or NA49 (10 μM) for 24 h, with or without gefitinib (10 or 20 μM) and NCI-H1975 cells were treated with J2 (10 μM) or NA49 (10 μM) for 24 h, with or without gefitinib (10 μM), and cell lysates were detected by Western blot. Protein levels were quantified using Image J software. The data were expressed as the fold change relative to the control and normalized to β-actin in the graph. (D) Cell death was analyzed by flow cytometry after propidium iodide (PI) staining. Results are the mean and standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated-control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. gefitinib alone 10 μM; + p < 0.05, ++ p < 0.01, +++ p < 0.001 vs. gefitinib alone 20 μM). (E) NCI-H1650 cells transfected with control (siCont) or siRNA of HSP27 (siHSP27) were treated with NA49 (10 μM) with or without gefitinib (10 μM), cell lysates were detected by Western blot analysis, and cell death was analyzed by flow cytometry after propidium iodide (PI) staining. Protein levels were quantified using Image J software. The data were expressed as the fold change relative to the control and normalized to β-actin in the graph. Results are the mean and standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated-control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. gefitinib alone).