Fig. 6
HMGB1 blockade by Gly attenuates MSI2-mediated CRC immunopathology in vitro and in vivo. A-B Western blot analysis of HMGB1 protein expression and qRT–PCR analysis of HMGB1, IL-6, TNF-a and IL-1b mRNA expression in stable SW620 and LOVO cells treated with DMSO or Gly (1 μM) for 12 h. C Diagram of the Gly (50 mg/kg) or PBS treatment protocol for CAC Msi2Transgenic mice. D Representative colon and spleen images of Gly blockade CAC models. Colon (top) and spleen (bottom). E Statistical analysis of colon length and spleen weight in Gly-treated CAC mice, n = 6. F HMGB1 mRNA expression was measured by qRT–PCR in Gly-treated CAC mice, n = 6. G Representative H&E, IHC and IFC images of HMGB1, CD3 and CD11c staining in Gly-treated CAC mice. Scale bars, 100 μm. H Inflammation score analysis for Gly-treated CAC mice, n = 6. I Statistical analysis of the CD3 average IHC staining density and the proportions of CD11c-positive cells. J Heatmap of different cytokines and chemokines levels in serum using Bio-Plex Pro Mouse Cytokine 23-Plex immunoassay from Gly-treated CAC mice, n = 6. K Statistical analysis of serum cytokines and chemokines in Gly-treated CAC mice, n = 6. L FACS and statistical analysis of the percentages of CD4+ T cells and CD8+ T cells isolated from MLNs of Gly- or PBS-treated CAC mice, n = 6. M FACS and statistical analysis of the percentages of CD4+ CD103+ T cells and CD8+CD103+ T cells isolated from MLNs of Gly- or PBS-treated CAC mice, n = 6. N The mRNA levels of colonic inflammation-associated genes were measured by qRT–PCR in Gly-treated CAC mice (n = 6). These results are presented as the mean ± SD values; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; B, E-F, H-I, K-M one-way ANOVA and N unpaired 2-tailed Student’s t test