Figure 1.

HBHA-induced apoptosis is associated with the ER stress response in macrophages. (a) Detection of apoptosis by cell cycle measurement. RAW 264.7 cells were stimulated with HBHA (10 μg/ml) for 24 h. The cells were stained with propidium iodide (PI) solution and the sub-G1 stage was determined by flow cytometry analysis. Exposure to 500 nM staurosporine (STS) for 6 h was used as positive control. (b) Immunoblot analysis of p-eIF2α, eIF2α, CHOP, GRP78, and β-actin in cell lysates of RAW 264.7 cells stimulated with HBHA (10 μg/ml) for the indicated time period. (c) Immunoblot analysis of CHOP, GRP78, p-eIF2α, caspase-3, and β-actin in BMDM cells stimulated with HBHA (10 μg/ml) for the indicated time period. Lysate of cells treated with 2 μg/ml tunicamycin (TM) for 6 h was used as a positive control. Bands corresponding to each protein were quantified, and the intensities of each protein were normalised to the intensity of actin. p-eIF2α was normalised to the intensity of eIF2α. Data are representative of at least three independent experiments with similar results. A significant difference was observed in HBHA-stimulated cells compared with unstimulated control (**P<0.01 and ***P<0.001). (d) Immunoblot analysis of HBHA, GRP78, and α-tubulin in both unstimulated control and HBHA-stimulated (10 μg/ml) cell lysates in RAW 264.7 cells. Subcellular fractionation was performed as described in the Materials and Methods