Fig 2: CUL3 promoted PD-L1 ubiquitination and degradation by forming a complex with SPOP in ovarian cancer cells. (A) Analysis of the expression of SPOP in ovarian cancer (normal group, n=38; cancer group, n=426) by gene expression profiling interactive analysis database. (B) Immunohistochemistry of SPOP protein expression in ovarian cancer tissue. (C) Detection of the mRNA expression of SPOP in adjacent normal tissues and ovarian cancer tissues (n=30) by RT-qPCR. (D) Detection of the interaction between PD-L1 and SPOP in SKOV3 cells by Co-IP experiment. (E) Co-IP experiment detected the interaction relationship between PD-L1 and SPOP in each group of SKOV3 cells. (F) Co-IP experiment detected the interaction relationship between SPOP and PD-L1. (G) RT-qPCR detection of the mRNA levels of SPOP and PD-L1 in SKOV3 cells after knocking down SPOP. (H) Western blot detection of the protein levels of SPOP and PD-L1 in SKOV3 cells after knocking down SPOP. (I) Co-IP experiment detected the interaction between PD-L1 and SPOP in SKOV3 cells after mutation of the binding site of SPOP and PD-L1. (J) Western blot detection of the protein degradation rate of PD-L1 in SKOV3 cells transfected with Flag-SPOP. **p<0.01, ***p<0.001. All experiment was repeated three times. Co-IP, co-immunoprecipitation; CUL3, cullin 3; mRNA, messenger RNA, PD-L1, programmed death ligand-1; RT-qPCR, reverse transcription quantitative PCR; SPOP, speckle type POZ protein.

Fig 3: Reduced CUL3 stabilized PD-L1 protein expression in ovarian cancer cells. (A) The correlation between CUL3 and PD-L1 (CD274) expression in ovarian cancer (n=307) was analyzed by tumor-immune system interactions database. (B) Reverse transcription quantitative PCR detection of CUL3 and PD-L1 mRNA levels after CUL3 overexpression in SKOV3 and A2780 cells. (C) Western blot detection of CUL3 and PD-L1 protein levels in SKOV3 and A2780 cells after CUL3 overexpression. (D) Western blot detection of CUL3 and PD-L1 protein levels in SKOV3 and A2780 cells after CUL3 knockdown. (E) Western blot detection of the effect of overexpressed CUL3 and MG132 and MLN4924 inhibitors on PD-L1 protein in SKOV3 and A2780 cells. (F) Co-IP experiment to detect the interaction between CUL3 and PD-L1 proteins in SKOV3 and A2780 cells. (G) Western blot was used to detect the effect of silencing CUL3 and CHX inhibitors on PD-L1 protein level in SKOV3 and A2780 cells. (H) Co-IP experiment to detect the interaction between CUL3 and PD-L1 ubiquitination protein after overexpression of CUL3 in SKOV3 and A2780 cells. **p<0.01, ***p<0.001, ns p>0.05. All experiments were repeated three times. Co-IP, co-immunoprecipitation; CUL3, cullin 3; mRNA, messenger RNA; PD-L1, programmed death ligand-1.

Fig 4: Schematic map of CUL3-mediated PD-L1 in ovarian cancer. CUL3 can form a complex with its linker molecule SPOP to promote the ubiquitination and degradation of PD-L1, leading to increased T-cell proliferation, activity and toxicity, thereby inhibiting the immune escape of ovarian cancer cells and improving the chemotherapy sensitivity of ovarian cancer cells. CUL3, cullin 3; IFN, interferon; IL, interleukin; PD-1, programmed cell death protein-1; PD-L1, programmed death ligand-1; SPOP, speckle type POZ protein.

Fig 5: Overexpression of CUL3 regulated the effect of PD-L1 expression on T cells and ovarian cancer cells. (A) RT-qPCR detection of CUL3 and PD-L1 mRNA levels in SKOV3 and A2780 cells after overexpression of CUL3 or PD-L1. (B) Western blot analysis detection of CUL3 and PD-L1 protein levels in SKOV3 and A2780 cells after overexpression of CUL3 or PD-L1. (C) Transwell for construction of a T cell, SKOV3 and A2780 cell co-culture system. (D) Detection of the proliferation of T cells (left) and SKOV3 (middle) and A2780 cells (right) in the co-culture system by CCK8. (E) Detection of the apoptosis rate of T cells in the co-culture system by flow cytometry. (F) Flow cytometry to detect the apoptosis rate of SKOV3 and A2780 cells in the co-culture system. (G) Lactate dehydrogenase release experiment detected the killing effect of T cells on SKOV3 and A2780 cells. (H) Detection of the changes in the mRNA levels of IL-2 and IFN-γ in each group of T cells in the co-culture system by RT-qPCR. (I) Detection of the changes in the protein levels of IL-2 and IFN-γ in the supernatant in the co-culture system by ELISA. **p<0.01, ***p<0.001. All experiments were repeated three times. CUL3, cullin 3; IFN, interferon; IL, interleukin; mRNA, messenger RNA; PD-L1, programmed death ligand-1; RT-qPCR, reverse transcription quantitative PCR.