Figure 4.

NGAL confers erlotinib resistance by enhancing ERK pathway activity and decreasing Bim protein levels. A: H3255 and H441 cells ectopically expressing NGAL (NGAL) and vector controls (V) were treated with erlotinib (0.1 μM for H3255 and 10 μM for H441) or vehicle control (DMSO) for 1-24 hours, as indicated. Cell lysates were analyzed for Erk pathway activation and Bim protein levels by Western blot. C - untreated cells in medium supplemented with 10% FBS. Cn - untreated cells in serum-free medium. B: NGAL overexpressing H3255 and H441 cells were treated with the MEK inhibitors U0126 (U, 10 μM) or PD98059 (PD, 40 μM) or the diluent (DMSO) for 24 hours. Cell lysates were analyzed for Erk pathway activation and Bim protein levels by Western blot. Ctrl-untreated cells. C: The same cells were treated with MEK inhibitor U0126 (U, 10 μM) or diluent (DMSO) for 4 hours, and the cell lysates were analyzed for phospho-Bim (pBim) and Bim extra-long isoform (Bim EL) levels by Western blot. D: NGAL overexpressing (NGAL) and vector control (Vector) H3255 and H441 cells were treated with emetine (E, 10 μM) or emetine plus MG132 (E+M, 100 μM) for 5 hours in serum-free medium. Cell lysates were analyzed for Bim isoform levels by Western blot. C - diluent (DMSO) treated control cells. E: H3255 and H441 cells were serum-depleted for 2 hours and treated with recombinant NGAL (105 ng/mL) in serum-free medium for 0.5 and 1 hour. Cell lysates were analyzed for Erk activation by Western blot. F: The cells were treated with recombinant NGAL (105 ng/mL) for 1-24 hours as indicated, and cell lysates were analyzed for Bim isoform levels by Western blot. Ctrl - IgG control.