Figure 4.

Leukocytes operate as transporter of dsHMGB1 in tumor microenvironment. (A) Representative images of immunohistochemical staining for CD45 (upper panel) and HMGB1 (lower panel) on tibialis anterior (TA) muscle sections from control (Ctrl) vs. Lewis lung carcinoma (LLC)-bearing mice. Scale bars, 50 μm. (B,C) Western blot probed with anti-CD45, anti-HMGB1, and anti-GAPDH antibodies in reducing conditions (B), and quantification of total CD45 and HMGB1 protein levels normalized on GAPDH (C) (n = 4 mice). In (B), spleen lysate (5 μg) was added as positive control for CD45 expression. (D) Western blot probed with anti-HMGB1 antibody in non-reducing conditions on tibialis anterior (TA) lysates from control or LLC-bearing mice. The upper and lower bands in non-reducing conditions correspond to the fully reduced-HMGB1 (frHMGB1) and the disulphide-HMGB1 (dsHMGB1) isoforms, respectively. (E) Percentage of HMGB1 redox isoforms expression. A.U. = arbitrary unit (n = 4 mice/group). (F) Immunohistochemical staining for CD45 (upper panel) and HMGB1 (lower panel) on tumoral sections from LLC-bearing mice. Scale bars, 50 μm. (G–J) Western blot probed with anti-CD45, anti-HMGB1, and anti-GAPDH antibodies in reducing conditions on LLC cells and tumoral masses isolated from mice injected with LLC cells (G), and quantification of total CD45 and HMGB1 protein levels normalized on GAPDH (H). (I) Western blot probed with anti-HMGB1 antibody in non-reducing conditions on LLC cells and tumoral masses isolated from mice injected with LLC cells. (J) Percentage of HMGB1 redox isoforms expression in LLC cultured cells and tumoral masses from LLC-injected mice (J). A.U. = arbitrary unit (n = 5 cell replicates and n = 4 mice for tumoral masses). Data represent the means ± SEM and statistical significance was calculated by Student T-test (C,H) and Two-way ANOVA (E,J). *P < 0.05; ****P < 0.0001.