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. 2020 Aug 6;11:1258. doi: 10.3389/fimmu.2020.01258

Figure 3.

Figure 3

S100A8 and S100A9 are required for the mitochondrial apoptosis pathway. (A) EoL-1 cells were incubated with S100A8 and S100A9 (10 μg/mL) for 24, 48, and 72 h. After cell lysis, the pro-caspase 9, cleaved caspase 9, pro-caspase 3, caspase 3, cleaved caspase 3, AIF, Mcl-1, Bcl-2, Bax, phospho-Bad, and Bad in the lysates were detected by Western blotting. (B,C) EoL-1 cells were pretreated with or without 0.5 μM MG132 (B) and 10 μM z-DEVD-fmk (C) for 1 h, followed by incubation with S100A8 and S100A9 (10 μg/mL) for 48 h. Levels of Mcl-1 in the cell lysates were detected by Western blotting. β-actin was used as an internal control. (D) EoL-1 cells were incubated with S100A8 and S100A9 (10 μg/mL) for 24, 48, and 72 h, and supernatants were collected at appropriate time points. EoL-1 cells were incubated with S100A8 and S100A9 (10 μg/mL) for 24, 48, and 72 h in the absence or presence of the supernatant (n = 3). Apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. *p < 0.05 and **p < 0.01 indicate a significant difference between the control and stimulator-treated groups.