Fig. 4. Liver injury exacerbated by gefitinib in combination with APAP might be related to Nrf2. (A-E, G) HL-7702 cells were treated with 10 μM gefitinib and/or 1.25 mM APAP for 24 h. (A) The expression levels of Nrf2 and ACTB were determined by western blot. (B) The transcription level of NFE2L2 was determined by qPCR. (C) The expression levels of Keap1 and ACTB were determined by western blot. All experiments were performed in triplicate. (D) The transcription levels of HO-1 and SOD-1 was determined by qPCR. (E) Representative images of HL-7702 stained with Nrf2 (green) and DAPI (blue) were shown by immunofluorescence assay. Scale bar=10 μm. (F) Induction of the ARE-dependent luciferase reporter activity by gefitinib and/or APAP. HL-7702 cells stably expressing an ARE-reporter gene were treated with 10 μM gefitinib and/or 1.25 mM APAP for 24 h and the luciferase activity in the cell lysates was measured. (G) The expression levels of p62 and ACTB were determined by western blot. All experiments were performed in triplicate. For western blots, one of three similar experiments is shown, and densitometric analysis was carried out. Data are presented as the mean ± SEM; n.s=no significance; *p<0.05; **p<0.01; ***p<0.001. APAP, acetaminophen; ARE, antioxidant response element; GEFI, gefitinib.
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