Journal: Oxidative Medicine and Cellular Longevity

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

doi: 10.1155/2022/8123157

Figure Lengend Snippet: The expression of STING in macrophages was induced in liver IRI.C57/B6 mice were subjected to liver IRI. (a) STING expression in the sham group and the IRI group in the presence or absence of liposomes (20 mM, 200 μ l/per mouse) was measured by Western blotting. (b) Relative expression of STING in each group. (c) STING expression in each group was measured by immunohistochemistry (scale bar, 50 μ m). (d) The colocalization of F4/80 and STING in KCs was measured by immunofluorescence in each group (scale bar, 100 μ m). All data are shown as the mean ± SD ( n = 6 mice per group). ∗∗∗ P < 0.001, ∗∗ P < 0.01, and ∗ P < 0.05.

Article Snippet: Frozen sections of liver tissue or KCs were next incubated in the dark at 4°C overnight with primary antibodies against STING (1 : 300, 19851-1-AP, Proteintech), F4/80 (5 μ g/mL, ab6640, Abcam), and caspase 1 (1 : 300, 14F468, Novus).

Techniques: Expressing, Western Blot, Immunohistochemistry, Immunofluorescence

Journal: Oxidative Medicine and Cellular Longevity

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

doi: 10.1155/2022/8123157

Figure Lengend Snippet: The expression of STING in liver macrophages was induced by hypoxia-reoxygenation (H/R) treatment. KCs were isolated from the liver and treated with H/R. (a) STING expression in the control group and the H/R group was measured by Western blotting. (b) Relative expression of STING in each group. (c) The mRNA levels of STING were measured by quantitative real-time PCR. (d) The colocalization of F4/80 and STING in KCs was measured by confocal laser scanning microscopy (CLSM) in each group (scale bar, 20 μ m). All data are shown as the mean ± SD ( n = 6 mice per group). ∗∗∗ P < 0.001, ∗∗ P < 0.01, and ∗ P < 0.05.

Article Snippet: Frozen sections of liver tissue or KCs were next incubated in the dark at 4°C overnight with primary antibodies against STING (1 : 300, 19851-1-AP, Proteintech), F4/80 (5 μ g/mL, ab6640, Abcam), and caspase 1 (1 : 300, 14F468, Novus).

Techniques: Expressing, Isolation, Western Blot, Real-time Polymerase Chain Reaction, Confocal Laser Scanning Microscopy

Journal: Oxidative Medicine and Cellular Longevity

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

doi: 10.1155/2022/8123157

Figure Lengend Snippet: Knockdown of STING in macrophages alleviated liver IRI. Mice were pretreated (i.v.) with AAV-STING-RNAi-F4/80-EGFP (1.5 × 10 11 vg) 14 days before liver IRI modeling. A volume of nonspecific AAV (AAV-Ctrl-F4/80-EGFP) equal to the treatment volume was administered in the same manner.(a) KCs were isolated from the liver in each group, and then the expression of STING in KCs in each group was measured by Western blotting. (b) Relative STING expression of KCs in each group. (c) STING expression in each group was measured by immunohistochemistry (scale bar, 200 μ m). (d, e) Serum levels of ALT and AST were measured. (f, g) H&E-stained sections of livers; average Suzuki scores were based on H&E-stained liver sections from different groups of mice (scale bar, 100 μ m). All data are shown as the mean ± SD ( n = 6 mice per group). ∗∗∗ P < 0.001, ∗∗ P < 0.01, and ∗ P < 0.05.

Article Snippet: Frozen sections of liver tissue or KCs were next incubated in the dark at 4°C overnight with primary antibodies against STING (1 : 300, 19851-1-AP, Proteintech), F4/80 (5 μ g/mL, ab6640, Abcam), and caspase 1 (1 : 300, 14F468, Novus).

Techniques: Isolation, Expressing, Western Blot, Immunohistochemistry, Staining

Journal: Oxidative Medicine and Cellular Longevity

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

doi: 10.1155/2022/8123157

Figure Lengend Snippet: Knockdown of STING in liver macrophages reduces caspase 1-GSDMD expression and H/R-induced inflammation. KCs were isolated from the liver and pretreated with STING-specific siRNA (20 μ M) or nonspecific siRNA (scramble) before H/R modeling. (a) The levels of STING, caspase 1, cleaved-caspase 1, GSDMD, and GSDMD-N in each group were measured by Western blotting. (b) The mRNA levels of STING were measured by quantitative RT-PCR. (c)–(g) Relative expression of STING, caspase 1, cleaved-caspase 1, GSDMD, and GSDMD-N in each group. (h) Caspase 1 activity was measured with a caspase 1 assay kit. (i) Supernatant LDH levels were measured. (j) The colocalization of caspase 1 and STING in KCs was measured by confocal laser scanning microscopy in each group (scale bar, 50 μ m). (k, l) The levels of cytokines (IL-1 β and IL-18) in the cell culture supernatant were measured by ELISA. All data are shown as the mean ± SD, ( n = 6). ∗∗∗ P < 0.001, ∗∗ P < 0.01, and ∗ P < 0.05.

Article Snippet: Frozen sections of liver tissue or KCs were next incubated in the dark at 4°C overnight with primary antibodies against STING (1 : 300, 19851-1-AP, Proteintech), F4/80 (5 μ g/mL, ab6640, Abcam), and caspase 1 (1 : 300, 14F468, Novus).

Techniques: Expressing, Isolation, Western Blot, Quantitative RT-PCR, Activity Assay, Confocal Laser Scanning Microscopy, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Oxidative Medicine and Cellular Longevity

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

doi: 10.1155/2022/8123157

Figure Lengend Snippet: Knockdown of STING reduces the intracellular calcium concentration in H/R-induced liver macrophages. KCs were isolated from the liver and pretreated with STING-specific siRNA (20 μ M) or nonspecific siRNA (Scramble) before H/R modeling. (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 μ m). (b) The relative concentration of calcium in each group was measured with a fluorescence microplate reader. All data are shown as the mean ± SD ( n = 6). ∗∗∗ P < 0.001, ∗∗ P < 0.01, and ∗ P < 0.05.

Article Snippet: Frozen sections of liver tissue or KCs were next incubated in the dark at 4°C overnight with primary antibodies against STING (1 : 300, 19851-1-AP, Proteintech), F4/80 (5 μ g/mL, ab6640, Abcam), and caspase 1 (1 : 300, 14F468, Novus).

Techniques: Concentration Assay, Isolation, Immunofluorescence, Labeling, Staining, Fluorescence

Journal: Oxidative Medicine and Cellular Longevity

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

doi: 10.1155/2022/8123157

Figure Lengend Snippet: 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 μ M) 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 μ m). (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-1 β 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 mean ± SD ( n = 6). ∗∗∗ P < 0.001, ∗∗ P < 0.01, and ∗ P < 0.05.

Article Snippet: Frozen sections of liver tissue or KCs were next incubated in the dark at 4°C overnight with primary antibodies against STING (1 : 300, 19851-1-AP, Proteintech), F4/80 (5 μ g/mL, ab6640, Abcam), and caspase 1 (1 : 300, 14F468, Novus).

Techniques: Isolation, Concentration Assay, Immunofluorescence, Labeling, Staining, Fluorescence, Western Blot, Quantitative RT-PCR, Expressing, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Transmission Assay, Electron Microscopy