Fig. 5

MSI2 directly increases disulfide HMGB1 production and translation by binding to nucleotides 1403–1409 in the 3’UTR of HMGB1. A Western blot analysis of disulfide and non-disulfide HMGB1 in lysates from stable SW620 and LOVO cells treated with or without DTT, and the Recombinant-HMGB1 (1 μg) incubated with either H₂O₂ or DTT (1 and 5 mM) were used as controls. B Exogenous Co-immunoprecipitation of Flag-MSI2 and Myc-HMGB1 in HEK 293 T cells. C Representative IFC images of MSI2 and HMGB1 localization in SW620 and LOVO cells. Scale bars, 20 μm. D MSI2 and HMGB1 protein levels in stable SW620 and LOVO cells at different time points after treatment with CHX (10 μM). E Reactome pathway enrichment analysis of the differentially expressed proteins identified by shotgun LC–MS/MS in HT29-OE/NC cells using the DAVID bioinformatics tool. F RIP assays were performed in LOVO and SW620 cells (anti-MSI2 and anti-IgG). G Detection of the HMGB1 gene by electrophoresis on a 2% agarose gel after RIP and qRT–PCR. H RNA pull-down assay was performed in LOVO cells, with Flowthrough supernatant (FT), biotinylated beads and lysate input as controls. I MSI2-specific RRMs predicted with beRBP: UAGUUAG. Binding scores (0.758 and 0.804) and putative positions of nucleotides 1403–1409 in the 3’UTR of HMGB1. J Wild-type (TAGTTAG) and mutant (AAAAAA) 3’UTRs of HMGB1 were inserted into the pmiR-GLO plasmid. K Firefly luciferase activity was measured in HEK 293 T cells transfected with MSI2-Flag and the wild-type or mutant pmiR-GLO plasmid and normalized to Renilla luciferase activity. These results are presented as the mean ± SD values; ****p < 0.0001; F unpaired 2-tailed Student’s t test and K one-way ANOVA