Journal: Clinical and Translational Medicine

Article Title: Ubc9‐mediated SUMOylation of Ninj1 alleviates inflammatory responses in hepatic ischaemia/reperfusion injury

doi: 10.1002/ctm2.70677

Figure Lengend Snippet: Blocking Hmgb1 reverses the aggravated injury of hepatocyte‐specific Ubc9 knockout in hepatic ischaemia/reperfusion (I/R)‐induced damage. (A) Schematic of anti‐Hmgb1 neutralisation experiments strategy in hepatic I/R. (B) Representative haematoxylin and eosin (H&E) staining images in livers from Ubc9 HKO mice treated with anti‐Hmgb1 antibody in hepatic I/R ( n = 8). Scale bar = 20 or 100 µm. (C) Quantification of necrotic areas in livers from Ubc9 HKO mice treated with anti‐Hmgb1 antibody in hepatic I/R ( n = 8). (D) The Suzuki scores of liver sections in Ubc9 HKO mice treated with anti‐Hmgb1 antibody in hepatic I/R ( n = 8). (E) Serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels in Ubc9 HKO mice treated with anti‐Hmgb1 antibody in hepatic I/R ( n = 8). (F) Immunohistochemical (IHC) staining of F4/80 + macrophages and Ly6G + neutrophils infiltration in livers from Ubc9 HKO mice treated with anti‐Hmgb1 antibody in hepatic I/R ( n = 8). Scale bar = 20 µm. (G) Serum levels of Il‐1β, Il‐6 and Tnf‐α were detected by ELISA in Ubc9 HKO mice treated with anti‐Hmgb1 antibody in hepatic I/R ( n = 8). (H) RT‐qPCR results for pro‐inflammatory genes ( Il‐1β , Il‐6 and Tnf‐α ) expression in livers from Ubc9 HKO mice treated with anti‐Hmgb1 antibody in hepatic I/R ( n = 8). (I) Schematic representation of co‐culture experiment for conditioned medium (CM) from hepatocytes to stimulate bone marrow‐derived macrophages (BMDMs), or CM plus anti‐Hmgb1 antibody to stimulate BMDMs. (J) Il‐1β, Il‐6 and Tnf‐ɑ in culture supernatants were measured by ELISA in BMDMs stimulated by CM from AML12 cells, or CM plus anti‐HMGB1 antibody ( n = 3). (K) RT‐qPCR analysis of Il‐1β , Il‐6 and Tnf‐ɑ in BMDMs stimulated by CM from AML12 cells, or CM plus anti‐Hmgb1 antibody ( n = 3). (L) Schematic representation of transwell experiment for CM from AML12 cells to stimulate BMDMs, or CM plus anti‐Hmgb1 antibody to stimulate BMDMs. (M) Transwell migration assay in BMDMs stimulated by CM from AML12 cells to stimulate BMDMs, or CM plus anti‐Hmgb1 antibody to stimulate BMDMs ( n = 3). (N) Western blot analysis of nuclear factor‐kappa B (NF‐κB) signalling pathway in BMDMs stimulated by CM from AML12 cells, or CM plus anti‐Hmgb1 antibody ( n = 3). Phosphorylated proteins (p‐P65 and p‐IKKβ) were normalised to relative total proteins (P65 and IKKβ). IκBɑ was normalised to β‐actin. IRI6h: ischaemia for 1 h followed by reperfusion for 6 h. All the data are presented as the mean ± SEM. One‐way analysis of variance (ANOVA) was used in (C–E), (G), (H), (J), (K), (M) and (N). ** p < .01; *** p < .001.

Article Snippet: Anti‐Hmgb1 neutralising (Arigo Biolaboratories, SQab20175) antibody, at a dosage of 100 μg dissolved in 100 μL sterile phosphate‐buffered saline (PBS), was administered intravenously through the tail vein 1 h before hepatic I/R.

Techniques: Blocking Assay, Knock-Out, Staining, Immunohistochemical staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Co-Culture Assay, Derivative Assay, Transwell Migration Assay, Western Blot