Fig. 2: Exogenous IL-26 activates bypass pathway of EGFR-TKI in mouse TNBC in in vitro assays.

A Phase contrast microscopy of E0771 cells (1 × 10⁴) following 48 h incubation with IL-26 or control vehicle in the presence or absence of gefitinib. Original magnification, ×100. Scale bar, 100 μm. Data shown are representative images of five independent experiments with similar results. B E0771 cells were treated with the indicated dose of IL-26 in the presence or absence of gefitinib (20 μM) for 48 h. *p < 0.01. C E0771 cells were treated with IL-26 (30 ng/ml) in the presence of various doses of gefitinib (5, 10, 15, or 20 μM). *p < 0.01. D E0771 cells were treated with IL-26 (30 ng/ml) and/or gefitinib (20 μM) in the presence of anti-IL-26 neutralizing mAb or isotype control mouse IgG (20 µg/ml, each) for 48 h. The dashed line is the standard value of gefitinib plus vehicle. E E0771 cells were stimulated with IL-26 (30 ng/ml) for the indicated periods, and then submitted to Western blot analysis using anti-phosphorylated AKT, JNK, ERK, p38, and STAT3 antibodies, and reblotting with anti-pan AKT, JNK, ERK, p38, and STAT3 antibodies. F E0771 cells were stimulated with IL-26 (30 ng/ml) in the presence or absence of gefitinib (20 μM) for 15 min, and then submitted to Western blot analysis as described in E. G E0771 cells were treated with IL-26 (30 ng/ml) and/or gefitinib (20 μM) in the presence or absence of various concentrations of signal inhibitors (AKT inhibitor, JNK inhibitor or combination of AKT inhibitor and JNK inhibitor) for 48 h. The dashed line is the standard value of gefitinib plus vehicle. *p < 0.01. B–D, G Cell proliferation was assessed by MTT assay. Representative data of five (B) and three (C, D, G) independent experiments are shown as mean ± S.D. of triplicate samples, and similar results were obtained in each experiment. E, F Data shown are representative of five independent experiments, and similar results were obtained in each experiment. Band intensity of phospho-proteins was normalized to the appropriate pan proteins, and relative intensity compared with unstimulated cells is shown as mean ± SEM from five independent experiments. *p < 0.01.