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. 2017 Feb 23;7:53. doi: 10.3389/fcimb.2017.00053

Figure 3.

Figure 3

The autophagy response induced by AFB1 was MEK/ERK-dependent and upregulated Beclin-1. (A,B) RAW264.7 cells and THP-1 cells were pretreated with Rapa (5 μM) for 12 h and then treated with AFB1 for different times. Western blotting was used for the Beclin-1 protein assay. The ratios of Beclin-1/GAPDH were calculated. (C) Western blotting of Beclin-1 in RAW264.7 cells with or without knockdown treatment was conducted. (D) RAW264.7 cells were transfected with the RFP-LC3 plasmid for 12 h and then transfected with the shRNA negative control or shBeclin-1 plasmids for 48 h before the cells were infected with AFB1 (0.25 μM) for 2 h. Fluorescence images show the induction of LC3 puncta. Scale bars = 20 μm. (E) The percentage of RFP-LC3 puncta cells was calculated. ***P < 0.001 compared with AFB1-infected cells; ###P < 0.001 compared with negative control shRNA plasmid. (F–H) RAW264.7 cells and THP-1 cells were pre-treated with the MEK/ERK inhibitor PD98059 (20 μM) for 1 h and then treated with AFB1 (0.25 μM) for P-ERK, P-MEK, and LC3 protein assays. (I) RAW264.7 cells were transfected with GFP-LC3 plasmid for 12 h. The cells were pretreated with PD98059 (20 μM, 1 h) and then treated with AFB1 (0.25 μM) for 2 h. Scale bars = 20 μm. (J) The percentage of GFP-LC3 puncta cells was calculated. ***P < 0.001 compared with the control groups; ###P < 0.001 compared with AFB1. (K) Western blotting of Beclin-1 was performed after treatment with PD98059 (20 μM, 1 h) and AFB1 (0.25 μM).