Fig 1: Dual mechanisms of cancer stemness regulation in osteosarcoma and targeted therapeutic strategies. (A) Dual mechanisms of ITGB2‐COPS3‐SOX2 axis and SOX2 liquid‐liquid phase separation in regulating cancer stemness in osteosarcoma. (B) Screening process for small‐molecule inhibitors targeting COPS3.
Fig 2: COPS3 stabilizes SOX2 by inhibiting its ubiquitination and promotes cancer stemness properties in osteosarcoma. (A–C) Interaction between COPS3 (yellow) and SOX2 (green) analyzed by PLIP. (D) Co‐immunoprecipitation verified COPS3 binding to SOX2. (E) Immunofluorescence staining verified the co‐localization of COPS3 with SOX2 (scale bar, 10 µm). (F) 143B cells with and without knockdown of COPS3 were subjected to CHX (50 µg/ml) assay. (G) Effect of the proteasome degradation inhibitor MG132 on SOX2 protein expression levels in COPS3 knockdown or non‐knockdown 143B cells. (H) The effect of COPS3 on SOX2 ubiquitination was analyzed after treatment with MG132 (20 µM) for 6 h. (I) COPS3 affected the expression of CSC‐related markers in Saos‐2 cells by affecting SOX2. (J) COPS3 affected ALDH activity in Saos‐2 cells by affecting SOX2, n = 5. (K) COPS3 affected the ability of Saos‐2 cells to resist anoikis by affecting SOX2, n = 5. (L) COPS3 affected the ability of Saos‐2 cells to resist doxorubicin by affecting SOX2, n = 5. (M) COPS3 affected the ability of Saos‐2 cells to sphere formation by affecting SOX2 (scale bar, 200 µm). (N) In vitro imaging of the SOX2 protein (scale bar, 10 µm). (O) Representative time‐lapse images showing the fission (split) and fusion (merge) events of SOX2 protein droplets in vitro. Red boxes highlight these dynamic processes, demonstrating the liquid‐like properties of the condensates. (P) Intracellular FRAP (scale bar, 5 µm). (Q) Immunofluorescence staining of CSC‐related markers in 143B cells after treatment with 1,6‐hexanediol (scale bar, 20 µm). ****p < 0.0001.
Fig 3: ITGB2 regulates stemness in osteosarcoma cells by affecting COPS3. (A‐B) Effect of knockdown of COPS3 on the ability of 143B and KHOS cells to adhere to vitronectin (A) and fibronectin (B). (C–E) Interaction between COPS3 (yellow) and ITGB2 (purple) analyzed by PLIP. (F) Co‐immunoprecipitation verified COPS3 binding to ITGB2. (G) Immunofluorescence staining verified the co‐localization of COPS3 with ITGB2 (scale bar, 20 µm). (H) Confocal microscopy showing that stimulation with the ITGB2 agonist Leukadherin‐1 (5 µM) promoted COPS3 nuclear accumulation in 143B cells (scale bar, 10 µm). (I) DEGs obtained by RNA sequencing between 143B cells with knockdown of ITGB2 and 143B cells without knockdown of ITGB2. (J) GO enrichment analysis of DEGs. (K) KEGG analysis of DEGs. (L) ITGB2 affected the expression of CSC‐related markers in Saos‐2 cells by affecting COPS3. (M) ITGB2 affected ALDH activity in Saos‐2 cells by affecting COPS3, n = 5. (N) ITGB2 affected the ability of Saos‐2 cells to resist anoikis by affecting COPS3, n = 5. (O) ITGB2 affected the ability of Saos‐2 cells to sphere formation by affecting COPS3 (scale bar, 200 µm). (P) ITGB2 expression in osteosarcoma primary tissues and osteosarcoma lung metastatic tissues (scale bar, 500 and 20 µm respectively). (Q) Colocalization analysis of COPS3 and ITGB2, as well as COPS3 and SOX2, in osteosarcoma primary tissues (scale bar, 10 µm). COPS3: green; ITGB2: red; SOX2: orange. Results are reported as means ± SD. ****p < 0.0001.
Supplier Page from Sino Biological, Inc. for Human SOX2 Protein (His Tag)