Fig. 4

MSI2 interacts with P300 to promote K29-HMGB1 acetylation, translocation and extracellular release. A Western blot analysis of acetyllysine in stable SW620 and LOVO cells. B Identification of lysine acetylation at K29 and K30 of HMGB1 by 4D label-free acetylome sequencing in stable SW620 cells. C Four gene clusters (Q1-Q4) based on the fold difference were identified in stable SW620 cells. D KEGG pathway enrichment analysis of the gene clusters (Q1-Q4) by BP and MF. E The DEGs identified by proteomics in stable SW620 and LOVO cells were subjected to KEGG pathway enrichment analysis using the DAVID bioinformatics tool. F Acetyl-K29-HMGB1 expression in the extracellular supernatant of stable SW620 and LOVO cells treated with or without 10 μg/mL LPS for 8 h. G Representative IFC images showing aberrant low expression of Acetyl-K29-HMGB1 in SW620 and LOVO cells with stable MSI2 knockdown. Scale bars, 100 μm. H-I Ectopic nucleocytoplasmic translocation of Acetyl-K29-HMGB1 in stable SW620 and LOVO cells in the absence (H) or presence (I) of LPS (10 μg/mL) for 8 h. J Acetyl-K29-HMGB1 and HMGB1 protein levels in HEK 293 T, SW620 and LOVO cells treated with TSA (1 μM) or (and) NAM (10 mM) for 12 h. K IP of HMGB1 and acetyllysine in stable SW620 and LOVO cells. L Positive correlations between MSI2 expression and KATs and KDACs expression in TCGA CRC database. M Heatmap of MSI2 and KAT/KDACs protein expression in stable SW620 cells as determined by 4D label-free proteomics. N MSI2, HDAC1, HDAC2, SIRT1 and EP300 mRNA expression was measured by qRT–PCR in stable SW620 and LOVO cells cultured in the presence of LPS (10 μg/mL) for 8 h. O Endogenous Co-immunoprecipitation of MSI2 and EP300 in SW620 and LOVO cells. P Representative IFC images of MSI2 and EP300 cytoplasmic localization in SW620 and LOVO cells. Scale bars, 10 μm. Q Representative IHC images of P300 from CAC mice and statistical analysis for P300 average IHC staining density. Scale bars, 100 μm. R Western blot analysis of EP300, HMGB1, K29-HMGB1 and NF-κB gene expression in CAC mice and stable CRC cells. These results are presented as the mean ± SD values; ns: not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001; N unpaired 2-tailed Student’s t test, Q Mann-Whitney test and L Pearson correlation analysis