Fig 2: Loss of MMS22L enhances chemosensitivity in SOX9-overexpressing CRC cells. (A,B) Immunofluorescence images showing the typical appearance (A) and percentages (B) of ?H2AX-positive cells in the indicated groups; nuclei were stained with DAPI (blue). Scale bar = 20 µm. (C,D) Flow cytometry results showing early apoptosis and the percentage of apoptotic cells among Vector+shCtrl, oeSOX9+shCtrl, Vector+shMMS22L, and oeSOX9+shMMS22L cells treated with oxaliplatin. **p < 0.01; ***p < 0.001; and ****p < 0.0001.

Fig 3: SOX9 enhances chemotherapeutic insensitivity in colorectal cancer by promoting MMS22L expression and nuclear translocation. (A) Representative images showing IHC staining of SOX9 and MMS22L in 2 CRC patients. Scale bar = 50 μm. (B) The correlation between SOX9 and MMS22L expression was analyzed in 80 CRC patients. (C,D) Representative immunofluorescence images showing SOX9 and MMS22L colocalization in CRC tissues and cell lines (scale bar = 20, 50, or 200 μm). (E) IP analysis of the interaction of MMS22L with SOX9 in SOX9-overexpressing RKO cells. (F) Immunofluorescence images showing the time-dependent variation in SOX9 and MMS22L colocalization in RKO cells treated with oxaliplatin. Scale bar = 20 μm. (G) Nuclear fractions were extracted from CRC cells, and the nuclear (neu) and cytoplasmic (without the nucleus; cyto) fractions were prepared. Immunoblot showing the protein levels of MMS22L and SOX9 expressed in Vector and oeSOX9 RKO cells and in shCtrl and shSOX9 RKO cells.

Fig 4: High expression of SOX9 promotes oxaliplatin resistance by inducing DNA repair in CRC cells. (A) Volcano plot showing the change in gene expression levels in shSOX9 RKO cells compared to control cells. (B) Heatmap showing the change in chemosensitivity and chemoresistance genes in shSOX9 RKO cells compared to control cells. (C,D) GSEA of drug sensitivity genes altered by SOX9 knockdown. (E,F) Representative images (E) and numbers (F) of early apoptotic shSOX9 and oeSOX9 CRC cells compared to control cells. (G) Cytotoxicity assay showing the proliferation of oeSOX9 and shSOX9 CRC cells treated with oxaliplatin compared to control cells. (H) GSEA of genes related to DNA damage altered by SOX9 knockdown in CRC cells. (I) Pathway enrichment analysis showing that the pathways related to DNA damage in shSOX9 CRC cells were extremely different from those in control cells. (J) Immunofluorescence showing representative images of ?H2AX (green) in oeSOX9 and shSOX9 CRC cells treated with oxaliplatin compared to control cells. Nuclei were stained with DAPI (blue) (scale bar = 20 µm). (K) Western blotting was used to detect the expression of MMS22L, NBS1, and ?H2AX in oeSOX9 and shSOX9 CRC cells treated with oxaliplatin compared to control cells. **p < 0.01; ***p < 0.001; and ****p < 0.0001.

Fig 5: Loss of MMS22L enhances chemosensitivity in SOX9-overexpressing CRC cells in vivo. (A–D) Antitumor outcomes of RKO xenografts in male nude mice treated with oxaliplatin, including photographs of the tumors (A), average tumor weights (B), and tumor volumes (C), at the end of the experimental period, along with the time-dependent variation curves of tumor volume (D). (E) IHC staining images showing the expression of SOX9, MMS22L, and ?H2AX in xenografts formed from Vector+shCtrl, oeSOX9+shCtrl, Vector+shMMS22L, and oeSOX9+shMMS22L cells. Scale bar = 50 µm. (F) Schematic diagram of the regulatory mechanism by which SOX9-mediated expression and nuclear translocation of MMS22L promotes DNA damage repair in CRC cells. **p < 0.01; ***p < 0.001; and ****p < 0.0001.