Journal: International Journal of Biological Sciences

Article Title: Atrial Natriuretic Peptide Attenuates Colitis via Inhibition of the cGAS-STING Pathway in Colonic Epithelial Cells

doi: 10.7150/ijbs.67356

Figure Lengend Snippet: Atrial natriuretic peptide (ANP) inhibits stimulator of interferon genes (STING) pathway activation and repairs gut barrier damage in a dextran sulfate sodium (DSS)-induced colitis mouse model. Mice with DSS-induced experimental colitis (n = 12 in each group) were intraperitoneally injected with ANP recombinant protein (2 μg in 400μL phosphate buffered saline [PBS] per mouse), DMXAA (0.1 mg in 400μL PBS per mouse) or 400μL PBS. Mice were sacrificed, and samples were collected on the 8th day. (A) Detailed method and dosing regimen for DSS, ANP and DMXAA. (B) Survival rates of mice with DSS-induced colitis. (C-F) Levels of STING pathway related proteins in colonic tissue, as identified by western blotting (n = 3 in each group). Representative blots are shown, and quantitative density data are shown as the intensity ratio of the target protein to relevant controls: cGAS/GAPDH; p-STING/STING; p-TBK1/TBK1; p-IRF3/IRF3. (G) Localization of E-cadherin (green fluorescence) and p-TBK1 (red fluorescence) within the colonic mucosa, as evaluated by immunofluorescence. (H) Localization of ZO-1 (green fluorescence) within colonic mucosa as evaluated by immunofluorescence. (I) Levels of ZO-1 and occludin in colonic tissue, as determined by western blotting (n = 5 in each group). Results were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (J) The levels of serum FD4 in different groups, reflecting colonic permeability. Statistical analysis was performed using one way analysis of variance (ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Compared with group treated with DSS and DMXAA, #P < 0.05).

Article Snippet: Meanwhile, prazosin (0.2 mg in 400 μL phosphate buffered saline [PBS] per mouse; Xinyi Pharmacy, China), human ANP recombinant protein (2 μg in 400 μL PBS per mouse; Tocris Bioscience, England), STING pathway agonist DMXAA (0.1 mg in 400 μL PBS per mouse; Topscience, China), or an equal volume of PBS as a control was injected intraperitoneally into the mice at the same time daily.

Techniques: Activation Assay, Injection, Recombinant, Saline, Western Blot, Fluorescence, Immunofluorescence, Permeability

Journal: International Journal of Biological Sciences

Article Title: Atrial Natriuretic Peptide Attenuates Colitis via Inhibition of the cGAS-STING Pathway in Colonic Epithelial Cells

doi: 10.7150/ijbs.67356

Figure Lengend Snippet: Atrial natriuretic peptide (ANP) promotes cGAS/NPR-A complex formation. HT-29 cells were treated with DMXAA or ANP 24 h in advance (n = 5 for each group). (A) Localization of NPR-A (green fluorescence) and cGAS (red fluorescence) within HT-29 cells, as evaluated by immunofluorescence. (B) HT-29 cells were subjected to immunoprecipitation with cGAS or NPR-A antibodies, followed by western blotting using the indicated antibodies.

Article Snippet: Meanwhile, prazosin (0.2 mg in 400 μL phosphate buffered saline [PBS] per mouse; Xinyi Pharmacy, China), human ANP recombinant protein (2 μg in 400 μL PBS per mouse; Tocris Bioscience, England), STING pathway agonist DMXAA (0.1 mg in 400 μL PBS per mouse; Topscience, China), or an equal volume of PBS as a control was injected intraperitoneally into the mice at the same time daily.

Techniques: Fluorescence, Immunofluorescence, Immunoprecipitation, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Atrial Natriuretic Peptide Attenuates Colitis via Inhibition of the cGAS-STING Pathway in Colonic Epithelial Cells

doi: 10.7150/ijbs.67356

Figure Lengend Snippet: Atrial natriuretic peptide (ANP) inhibits stimulator of interferon genes (STING) pathway activation and repairs gut barrier damage in colonic epithelial cells (CECs). HT-29 cells were treated with DMXAA or ANP 24 h in advance (n = 5 for each group). (A) Relative expression of interferon α (IFN-α), IFN-β, IFN-γ, cGAS, TBK1, and IRF3 in HT-29 cells was tested using quantitative real-time polymerase chain reaction (qRT-PCR. Results were normalized against the glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) gene. (B-E) Expression levels of STING pathway related proteins in colon tissue, as identified by western blotting. Representative blots are shown, and quantitative density data are shown as the intensity ratio of the target protein to relevant controls: cGAS/GAPDH; p-STING/STING; p-TBK1/TBK1; and p-IRF3/IRF3. (F) Protein levels of ZO-1, E-cadherin, and occludin in HT-29 cells, as determined by western blotting (n = 5 for each group). Results were normalized against GAPDH. (G) Localization of E-cadherin (green fluorescence) and p-TBK1 (red fluorescence) within HT-29 cells, evaluated by immunofluorescence. (H) Localization of ZO-1 (red fluorescence) within HT-29 by immunofluorescence.

Article Snippet: Meanwhile, prazosin (0.2 mg in 400 μL phosphate buffered saline [PBS] per mouse; Xinyi Pharmacy, China), human ANP recombinant protein (2 μg in 400 μL PBS per mouse; Tocris Bioscience, England), STING pathway agonist DMXAA (0.1 mg in 400 μL PBS per mouse; Topscience, China), or an equal volume of PBS as a control was injected intraperitoneally into the mice at the same time daily.

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Fluorescence, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: Atrial Natriuretic Peptide Attenuates Colitis via Inhibition of the cGAS-STING Pathway in Colonic Epithelial Cells

doi: 10.7150/ijbs.67356

Figure Lengend Snippet: Atrial natriuretic peptide (ANP) inhibits endoplasmic reticulum (ER) stress-induced autophagy in colonic epithelial cells (CECs) via stimulator of interferon genes (STING) pathway activation. HT-29 were treated with DMXAA or ANP 24 h in advance (n = 5 for each group). (A-B) Relative expression of Atg5, Atg7, and Atg12 in primary CECs extracted from mice and in HT-29 cells. Results were normalized against the β-actin/ glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) genes. (C) Expression levels of autophagy-related (LC3, free Atg12, Atg12-Atg5, Beclin1, P62) and ER stress-related (eIF2α, SAPK/JNK, BiP) proteins in HT-29 were identified using western blotting (n = 5 for each group). Representative blots are shown, and quantitative density data are shown as the intensity ratio of the target protein to relevant controls: LC3-II/LC3-I, Atg12-Atg5/GAPDH, Beclin1/GAPDH, P62/GAPDH, p-eIF2α/eIF2α, p- SAPK/JNK/SAPK/JNK, and Bip/GAPDH. (D-E) Localization of P62 (green fluorescence) in HT-29 cells, as evaluated by immunofluorescence. (F-H) The accumulation of StubRFP-SensGFP-LC3 in HT-29 cells treated with DMXAA or ANP was observed using confocal micrographs. Red arrows indicate autophagosomes and yellow arrows indicate autolysosomes.

Article Snippet: Meanwhile, prazosin (0.2 mg in 400 μL phosphate buffered saline [PBS] per mouse; Xinyi Pharmacy, China), human ANP recombinant protein (2 μg in 400 μL PBS per mouse; Tocris Bioscience, England), STING pathway agonist DMXAA (0.1 mg in 400 μL PBS per mouse; Topscience, China), or an equal volume of PBS as a control was injected intraperitoneally into the mice at the same time daily.

Techniques: Activation Assay, Expressing, Western Blot, Fluorescence, Immunofluorescence

Journal: Science immunology

Article Title: Intratumoral activation of the necroptotic pathway components RIPK1 and RIPK3 potentiates anti-tumor immunity

doi: 10.1126/sciimmunol.aaw2004

Figure Lengend Snippet: (A) Day 12 volumes of B16.F10-OVA tumors in various innate immune knockout mice following necroptotic fibroblast injections on days 6, 8, and 10. N=5–15 mice per group. (B) B16.F10-OVA tumor growth (left panel) and animal survival (right panel) upon co-administration of necroptotic fibroblasts with blocking α-CLEC9A antibody. N=10 mice per group. (C-D) Tumor growth and overall survival following administration of lytic necrotic fibroblasts in (C) single, or (D) contralateral B16.F10-OVA flank tumors. Tumor growth and survival curves for PBS, acCASP8, and acRIPK3 as presented in Figure 1 are also graphed for comparison. (E) B16.F10-OVA tumor growth curves following injection of necroptotic NIH-3T3 fibroblasts pre-incubated with the IκBα phosphorylation inhibitor BAY-117085, which prevents NF-κB activation in treated cells. N=10 mice per group. B16.F10-OVA tumor growth curves following intratumoral (IT), intraperitoneal (IP), distal subcutaneous (distal subQ), or intravenous (IV) injection of necroptotic fibroblasts. N=7–9 mice per group. (F) B16.F10-OVA tumor growth (left panel) and animal survival (right panel) following IT injection of PBS, necroptotic fibroblasts, or MLKL−/− necroptotic fibroblasts. N=7–9 mice per group. (G) Assessment of systemic inflammation via Luminex assay for inflammatory serum cytokines and chemokines 48h post-intratumoral dying NIH-3T3 injection. DMXAA-injected mice were included as a positive control for systemic inflammatory cytokine production. Gray dotted line represents limit of detection. N=3–5 mice per group. (H) B16.F10-OVA tumor growth curves following intratumoral (IT), intraperitoneal (IP), distal subcutaneous (distal subQ), or intravenous (IV) injection of necroptotic fibroblasts. N=7–9 mice per group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Black arrows indicate intratumoral dying cell injections. Error bars represent SEM. Data are pooled from 2–5 independent experiments.

Article Snippet: As a positive control for systemic inflammation, B6/J mice were injected intraperitoneally with 40mg/kg of the STING agonist DMXAA (ApexBio), and sera were harvested 5h post-injection, then frozen at −80°C.

Techniques: Knock-Out, Blocking Assay, Comparison, Injection, Incubation, Phospho-proteomics, Activation Assay, IV Injection, Luminex, Positive Control