Figure 6.

PLK1 is involved in the gefitinib-induced autophagy. (A) The transcription level of PLK1 was determined by qPCR (n = 3, one-way ANOVA, LSD test). (B) HL-7702 cells or human primary hepatocytes were treated with gefitinib. The expression levels of PLK1 and GAPDH were determined by western blot. (C–F) HL-7702 cells were transfected with non-targeting siRNA (NC) or siRNA targeting PLK1 (siPLK1) or COX6A1 (siCOX6A1), followed by treatment with or without gefitinib. HL-7702 cells were transfected with pCMV6-PLK1 or pcDNA3.0-COX6A1 or vector, followed by treatment with or without gefitinib for 24 h. (C) The expression levels of PLK1, LC3, COX6A1 and c-PARP were measured by western blot. (D, E) Representative confocal fluorescence micrographs of HL-7702 cells transfected with Ad-mCherry-GFP-LC3B. Scale bar: 20 μm. Quantification of autophagosome and autolysosome puncta was represented (n > 30, one-way ANOVA, Dunnett T3 test). (F) Apoptotic cells were quantified by flow cytometry with ANXA5-PI staining (n = 3, one-way ANOVA, LSD test). Western blot was repeated at least three times and densitometric analysis was carried out. The data are expressed as the mean ± SD; n.s = no significance; * p < 0.05; **p < 0.01; ***p < 0.001. Abbreviations: GEFI, gefitinib; c-PARP, cleaved PARP