Fig. 5

MSI2 deficiency triggers CRC ferroptosis by downregulating the MAPK signaling cascade to inhibit HSPB1 phosphorylation. A The functions of genes corresponding to differentially expressed proteins were annotated based on KOG/COG, and the main enriched metabolic pathways were lipid transport and metabolism and inorganic ion transport and metabolism. B-C Genes corresponding to the differentially expressed proteins were functionally annotated based on the KEGG database and were mainly enriched in nonalcoholic fatty liver disease, oxidative phosphorylation, chemical carcinogenesis-reactive oxygen species and MAPK signaling pathway. D Circular heatmap demonstrating the most significant differential expression of ferroptosis-related genes in stable SW620 cell proteomics analysis. E Heatmap showing the differential expression of ferroptosis-related driver or suppressor genes in stable SW620 cell proteomics analysis. F Verification of differential gene expression in SW620, LOVO, and HT29 stable cells by using Western blotting for MSI2, HSPB1, p-HSPB1(Ser78), ACSL4, FTH1, TFRC and GPX4. G Representative IFC images of p-HSPB1(Ser78) expression in SW620, LOVO and HT29 stable cells. Blue: DAPI, red: p-HSPB1(Ser78); Scale bars, 50 μm. H HSPB1, ACSL4, FTH1 and GPX4 genes mRNA expression were determined by qRT‒PCR in SW620, LOVO, and HT29 stable cells. I Co-immunoprecipitation assays showed that there was no direct interaction between MSI2 and HSPB1 in SW620 and LOVO cells. FT means flow through wash fraction, input and anti-IgG as controls. J The protein–protein interaction (PPI) networks between HSPB1 and MAPK family genes were constructed by STRING. K Differential MAPK signaling pathway genes expression in stable SW620 cell proteomics analysis. L Co-immunoprecipitation assays showed that MSI2 could interact with p-ERK(p-P42/44) in SW620, LOVO and HEK293T cells. The input and anti-IgG as controls. M The protein levels of p-ERK(p-P42/44) were determined by IFC in SW620, LOVO and HT29 stable cells. Blue: DAPI, green: p-ERK(p-P42/44); Scale bars, 50 μm. N Western blotting for MAPK signaling cascade gene expression, such as ERK(P42/44), p-ERK(p-P42/44), P38, p-P38, MAPKAPK2, MAPK13, p-MAPKAPK2 and PCNA, in SW620, LOVO, and HT29 stable cells. These results are presented as the mean ± SD values; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; H unpaired 2-tailed Student’s t test