Fig. 2. APAP enhanced gefitinib-triggered apoptosis. (A) Cell proliferation inhibition was examined by the SRB assays. HL-7702 cells were exposed to gefitinib, APAP and/or different inhibitors: 20 μM Z-VAD-FMK, 10 μM Z-YVAD-FMK, 1μM ferrostatin-1, 20 μM necrostatin-1 and 1mM 3-MA for 48 h. (B-C) HL-7702 cells were treated with 10 μM gefitinib and/or 1.25 mM APAP for 24 h. (B) The apoptosis rates were analyzed by flow cytometry with an FITC Annexin V Apoptosis Detection Kit. (C) The expression levels of c-PARP and ACTB were determined by western blot. (D) Liver sections were stained with TdT-mediated dUTP nick end labeling (TUNEL) and 4’,6-diamidino-2-phenylindole (DAPI). Scale bar=50 μm. Quantitative analysis was performed to detect apoptotic cells (n=3). (E) HL-7702 cells treated with 10 μM gefitinib and 1.25 mM APAP were combined with or without 20 μM Z-VAD-FMK for 24 h. The expression levels of c-PARP 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=3); n.s=no significance; *p<0.05; **p<0.01; ***p<0.001. APAP, acetaminophen; Fer-1, ferrostatin-1; GEFI, gefitinib; Nec-1, necrostatin-1; SRB, sulforhodamine B; 3-MA, 3-Methyladenine.
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