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. 2015 Aug 20;16(8):19780–19795. doi: 10.3390/ijms160819780

Figure 5.

Figure 5

Effect of CHOP on the ZEA-induced cell death of RAW 264.7 macrophages. RAW 264.7 macrophages were transduced with CHOP or non-targeting lentiviral-mediated shRNAs. (A) The constructs themselves could express GFP, and the proportion of cells that were transduced was 90% by flow cytometry (data not shown). Fluorescence images of RAW 264.7 macrophages transducted for 48 h with negative control shRNA (shNC) (a), shCHOP (b) and control (c) virus-containing supernatant; GFP expression was observed under light (top panels) or fluorescence microscopy (bottom panels). Bars = 50 µm; (B,C) The expression levels of CHOP were determined by western blot analysis. RAW 264.7 macrophages were transduced with lentiviruses expressing either shCHOP or shNC, and were then treated with 30 μM ZEA for 24 h. The analyses of the band intensity on the films are presented as the relative ratio of CHOP to β-actin. Statistical analysis is shown in the bar graphs; (D) Transduced RAW 264.7 macrophages with lentiviruses expressing either shCHOP or shNC were treated with 0, 30 and 50 μM ZEA and then cell viability was measured after 24 h by the MTT assay; (E,F) RAW 264.7 macrophages were tranduced with shNC or shCHOP to confirm the effects of shCHOP. To determine the apoptotic effect of ZEA on the RAW 264.7 macrophages, cells were transduced with shNC or shCHOP for 48 h and then incubated for 24 h in the absence or presence of 30 μM ZEA. Apoptosis was determined by Annexin V-PE/7-AAD staining. B1 is the part of cell death caused by mechanical damage, B2 is the part of late apoptotic or necrotic cells, B3 is the part of the normal cells, and B4 is the part of early apoptotic cells. Statistical analysis is shown in the bar graphs. Data are presented as the mean ± SEM of three independent experiments. Bars with different letters are significantly different (p < 0.05).