Journal: bioRxiv
Article Title: MG53 Protects Against Intestinal Inflammation by Inhibiting NLRP3 Inflammasome Activation
doi: 10.64898/2026.02.18.706669
Figure Lengend Snippet: A . Immunoblot of IL-1β p17 and caspase-1 p20 in supernatants (Sup.) from lipopolysaccharide (LPS)-primed mouse bone marrow–derived macrophages (BMDMs) (100 ng/ml, 3 h) treated with rhMG53 for 1 h and stimulated with nigericin for 1 h. Pro–IL-1β, pro–caspase-1, NOD-like receptor family pyrin domain containing 3 (NLRP3), and MG53 were analyzed in cell lysates. B . IL-1β in culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA). Treatments as in ( A ). n=3. C . Immunoblot of IL-1β p17 and caspase-1 p20 in supernatants from LPS-primed BMDMs treated with rhMG53 (10 μg/ml, 3 h) and stimulated with nigericin (10 μg/ml, 1 h), adenosine 5′-triphosphate disodium salt (ATP) (5 mM, 30 min), or monosodium urate crystals (MSU) (150 μg/ml, 5 h). Pro–IL-1β, pro– caspase-1, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC) and MG53 were assessed in lysates. D . ELISA of IL-1β release in culture supernatants. Treatments as in ( C ). n=3. E. BMDMs were treated with rhMG53 (1, 5, 10 μg/ml) for 1h before or after LPS stimulation, and NLRP3 inflammasome-related proteins were examined by immunoblotting. F . Immunoblot of IL-1β p17 and caspase-1 p20 in culture supernatants of LPS-primed WT or MG53-overexpressing transgenic mice (TPA) BMDMs treated for 4 h and stimulated with nigericin (10 μg/ml, 1 h) or ATP (5 mM, 30 min). MG53, NLRP3, ASC, pro-IL-1β, and pro-caspase-1 were analyzed in cell lysates. G . ELISA of IL-1β release in supernatants from LPS-primed WT or TPA BMDMs treated for 3 h and stimulated with nigericin (10 μg/ml,1h) or ATP (5 mM, 30 min). n=5. H . THP-1 cells differentiated with PMA were primed with LPS (0.5 μg/ml, 4 h), treated with rhMG53 at the indicated concentrations for 1 h, and stimulated with nigericin (1 μg/ml, 2 h). Supernatants and cell lysates were analyzed by immunoblotting. IL-1β p17 and caspase-1 p20 were detected in supernatants, while pro-caspase-1, pro-IL-1β, NLRP3, ASC, and MG53 were detected in lysates. I . ELISA measurement of IL-1β release in culture supernatants. n=3. Data are mean ± SD. Statistical significance was determined by one-way ANOVA ( B and I ) and unpaired Student’s t-test ( D and G ), *** p < 0.005, ** p < 0.01, * p < 0.05.
Article Snippet: Lipopolysaccharide (LPS) from Escherichia coli 0111: B4 (tlrl-eblps), Nigericin sodium salt (tlrl-nig), adenosine 5′-triphosphate disodium salt (ATP, tlrl-atpl) and monosodium urate crystals (MSU, tlrl-msu) were from InvivoGen.
Techniques: Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay, Transgenic Assay