STING Induces Liver Ischemia-Reperfusion Injury by Promoting Calcium-Dependent Caspase 1-GSDMD Processing in Macrophages

Figure 7

H/R-induced STING increases intracellular calcium to promote caspase 1-GSDMD processing in liver macrophages. KCs were isolated from the liver and treated with H/R in the absence or presence of BAPTA-AM (10 μM) for 24 h.(a) The concentration of calcium was measured by immunofluorescence, where intracellular calcium was labeled with Fluo-4 AM (green), and the nucleus was labeled with Hoechst stain (scale bar, 100 μm). (b) The relative concentration of calcium in each group was measured with a fluorescence microplate reader. (c) The levels of STING, caspase 1, cleaved-caspase 1, GSDMD, and GSDMD-N in each group were measured by Western blotting. (d) The mRNA levels of STING were measured by quantitative RT-PCR. (e)–(i) Relative expression of STING, caspase 1, cleaved-caspase 1, GSDMD, and GSDMD-N in each group. (j) Caspase 1 activity was measured with a caspase 1 assay kit. (k) Supernatant LDH levels were measured. (l, m) The levels of cytokines (IL-1β and IL-18) in the cell culture supernatant were measured by ELISA. (n) Transmission electron microscopy (TEM) was used to observe the ultrastructural changes in KCs (original magnification, ×20000). The red arrow indicates the incomplete structure of an organelle, and the blue arrow indicates discontinuity in the cell membrane. All data are shown as the (). , , and .

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