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. 2023 Feb 22;13(3):415. doi: 10.3390/biom13030415

Figure 5.

Figure 5

APOL6 induced immunogenic cell death in vitro. (A) Immunoblot analysis of FLAG-tagged APOL6 expression in transfected MIA PaCa-2 cells. (B) Cell viability of APOL6-transfected MIA PaCa-2 cells was determined using CCK8 assay. MIA PaCa-2 cells were seeded in 96-well plates and transfected with various doses of APOL6 overexpression plasmid for the indicated time-points. The APOL6 protein amount in MIA PaCa-2 cells transfected with APOL6 expression plasmids at different doses was shown in Figure S2. (C) Cell death of APOL6-transfected MIA PaCa-2 cells was detected with PI staining by flow cytometry. (D) The expression of cytokines and chemokines involved in type I interferon response was measured by qRT-PCR. (E) APOL6-transfected MIA PaCa-2 cells were treated with Z-VAD-FMK (10 µM) [23], necrostatin-1 (10 µM) [23], VX765 (10 µM) [22] and ferrostatin-1 (2 µM) [23] for 72 h, and cell death was evaluated with PI staining by flow cytometry. Except where noted, FLAG-tagged APOL6 expression plasmids were transfected in 6-well plates at doses of 5000 ng. The p values were calculated using the t-test. NC, negative control. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.