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. 2020 Jun 24;11:1122. doi: 10.3389/fimmu.2020.01122

Figure 1.

Figure 1

HMGB1 redox isoforms expression and leukocyte infiltration during acute muscle injury. (A) Representative images of immunohistochemical staining for CD45 (upper panel) and HMGB1 (lower panel) on tibialis anterior (TA) muscle sections at indicated time points after cardiotoxin (CTX) injection. Scale bars, 50 μm. Ctrl, uninjured control muscles. (B–E) Western blot probed with anti-CD45 (upper panel) and anti-HMGB1 (middle panel) antibodies in reducing conditions or with anti-HMGB1 antibody in non-reducing conditions (lower panel) on muscle lysates at indicated time points after CTX injection. The upper and lower bands in non-reducing conditions correspond to the fully reduced-HMGB1 (frHMGB1) and the disulphide-HMGB1 (dsHMGB1) isoforms, respectively. (C) Quantification of total HMGB1 protein expression levels, relative to control (Ctrl) and normalized on Ponceau staining, at indicated time points after CTX injection. A.U. = arbitrary unit (n ≥ 10 muscles, 3 mice/time point). (D) Quantification of CD45 and HMGB1 redox isoforms expression (frHMGB1 and dsHMGB1), normalized on Ponceau staining, at indicated time points. (E) Distribution of HMGB1 redox isoforms expression in muscle lysates in absence (controls and at 1 h post-injury) or presence of CD45-positive cells (from 6 h to day 7 post-injury). (F–I) Western blot probed with anti-HMGB1 antibody in reducing (upper panel) and non-reducing conditions (lower panel) on supernatant of muscles isolated at indicated time points after CTX injection (F). Total HMGB1 protein expression at indicated time points after CTX injection (G). A.U. = arbitrary unit (n = 6 muscle supernatants, 3 mice/time point). (H) Quantification of HMGB1 redox isoforms expression (frHMGB1 and dsHMGB1) in muscle supernatants, from Western blot assays in non-reducing conditions, at indicated time points after CTX injection and compared with CD45 expression as in (D). (I) Distribution of HMGB1 redox isoforms expression in supernatants of muscle in absence (controls and at 1 h post-injury) or presence of CD45-positive cells (from 6 h to day 7 post-injury). Data represent the means ± SEM and statistical significance was calculated by One-way (C,D,G,H) and Two-way ANOVA (E,I). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.