Fig 2: The mechanism of ASPA in PCa. ASPA aspartoacylase, AP-1 Activator protein-1, C-Jun v-Jun avian sarcoma virus 17 oncogene homolog, JNK c-Jun N-terminal kinase, LYN Lck/Yes-related novel protein tyrosine kinase, PCa prostate cancer

Fig 3: ASPA interacts with the N-terminus of LYN and regulates its activity in an enzyme-independent manner. a The interaction domain(s) between ASPA (left) and LYN (right) were determined by IP assays in HEK293T cells using full-length and truncated LYN or ASPA expression constructs. b Western blotting analysis (top) and quantification (bottom) of the phosphorylation of LYN, JNK1/2, and C-Jun in PC-3 cells transfected with the Flag-ASPA and Flag-ASPA ?1-212 (deletion of amino acids 1-222) vectors. Protein expression levels were normalized to ß-actin levels. c The proliferation ability of PC-3 cells transfected with Flag-ASPA and Flag-ASPA ?1-212 vectors was assessed using a CCK-8 assay. d Cell colony formation ability (top) and migration ability (bottom) of PC-3 cells transfected with Flag-ASPA and Flag-ASPA ?1-212 vectors were assessed using colony formation and Transwell assays. The graph on the right shows the colony numbers (left) from 3 independent experiments and the number of migrated cells (right) obtained from 8 fields of 3 independent experiments (scale bar = 100 µm). e Co-IP assays showed that the interaction between ASPA and LYN was not affected by the E178D mutation in HEK293T cells cotransfected with HA-LYN, Flag-ASPA and Flag-ASPA E178D vectors. f Western blotting analysis (left) and quantification (right) of ASPA and the phosphorylation of LYN, JNK1/2, and C-Jun in PC-3 cells transfected with the Flag-ASPA and Flag-ASPA E178D vectors. Protein expression levels were normalized to ß-actin levels. g The proliferation ability of PC-3 cells transfected with Flag-ASPA and Flag-ASPA E178D vectors was assessed using a CCK-8 assay. h Cell colony formation ability (top) and migration ability (bottom) of PC-3 cells transfected with Flag-ASPA and Flag-ASPA E178D vectors were assessed using colony formation and Transwell assays. The graph below shows the colony numbers (left) from 3 independent experiments and the number of migrated cells (right) obtained from 8 fields of 3 independent experiments (scale bar = 100 µm). i The mechanism of ASPA in PCa. The data are presented as the mean ± standard deviation (SD). ASPA aspartoacylase, CCK-8 cell counting kit 8, C-Jun v-Jun avian sarcoma virus 17 oncogene homolog, Co-IP coimmunoprecipitation, HEK-293 T human embryonic kidney cell 293 T, JNK c-Jun N-terminal kinase, LYN Lck/Yes-related novel protein tyrosine kinase, OD optical density, PCa prostate cancer. **P < 0.01, ns not significant

Fig 4: LYN mediates the function of ASPA in PCa cells. a Western blotting assay showed ASPA expression and the activity of LYN, JNK1/2, and C-Jun in PC-3 cells cotransfected with ASPA overexpression vector and/or LYN overexpression vector. b The cell proliferation ability of PC-3 cells cotransfected with ASPA overexpression vector and/or LYN overexpression vector was assessed using CCK-8 assay. c Western blotting assay showed ASPA expression and the activity of LYN, JNK1/2, and C-Jun in PC-3 cells transfected with shASPA and/or treated with the LYN inhibitor bafetinib. d The proliferation ability of PC-3 cells transfected with shASPA and/or treated with bafetinib was assessed using a CCK-8 assay. e Representative images of subcutaneous xenograft tumors derived from PC-3 cells transfected with shASPA and/or treated with bafetinib. f The volume of xenograft tumors derived from PC-3 cells transfected with shASPA and/or treated with bafetinib (n = 8). g The weight of xenograft tumors derived from PC-3 cells transfected with shASPA and/or treated with bafetinib (n = 8). h RT-qPCR results of proliferation-related genes and epithelial–mesenchymal transition genes in xenograft tumors derived from PC-3 cells transfected with shASPA and/or treated with bafetinib. The mRNA expression levels were normalized to ACTB levels. i Western blotting analysis (left) and quantification (right) of ASPA and the phosphorylation of LYN, JNK1/2, and C-Jun in subcutaneous xenograft tumors derived from PC-3 cells transfected with shASPA and/or treated with bafetinib. Protein expression levels were normalized to ß-actin levels. The data are presented as the mean ± standard deviation (SD). ASPA aspartoacylase, CCND1 cyclin D1, CDH1 Cadherin 1, CCK-8 cell counting kit 8, C-Jun v-Jun avian sarcoma virus 17 oncogene homolog, DMSO dimethyl sulfoxide, JNK c-Jun N-terminal kinase, LYN Lck/Yes-related novel protein tyrosine kinase, OD optical density, RT-qPCR real-time quantitative PCR, PCNA proliferating cell nuclear antigen, shRNA small hairpin RNA. *P < 0.05, **P < 0.01, ns not significant