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: ASPA directly interacts with LYN and suppresses its phosphorylation. a Flow chart of the mass spectrometry analysis of ASPA-overexpressing PC-3 cells and control cells. b Five overlapping proteins were identified between ASPA binding proteins (306) and kinase proteins (511) in HEK293T cells. c Co-IP analysis of the interaction between ASPA and the indicated Flag-tagged five kinase proteins in PC-3 cells. d Exogenous IP assays were performed to evaluate the binding of ASPA and LYN in PC-3 cells transfected with Flag-LYN or Flag-ASPA. e Endogenous IP assays were performed in HEK293T cells transfected with Flag-LYN or HA-ASPA. f GST pull-down assays showed the direct binding of ASPA to LYN using GST-HA-ASPA and Flag-LYN (left) or using GST-HA-LYN and Flag-ASPA (right). g The colocalization of ASPA and LYN in PC-3 cells was determined by immunofluorescence (scale bar = 100 µm). h Western blotting results (left) and quantification results (right) of the phosphorylation of LYN in PC-3 cells transfected with control and ASPA overexpression vector or shRNA and shASPA. Protein expression levels were normalized to ß-actin levels. i Western blotting results (left) and quantification results (right) of the phosphorylation of LYN in xenograft tumors derived from PC-3 cells transfected with control or ASPA overexpression vector or shRNA and shASPA. Protein expression levels were normalized to ß-actin levels. j Western blotting results (left) and quantification results (right) of ASPA and the phosphorylation of LYN in 6 PCa samples with paired normal tissues. Protein expression levels were normalized to ß-actin levels. The data are presented as the mean ± standard deviation (SD). ASPA aspartoacylase, Co-IP coimmunoprecipitation, CSNK2A1 casein kinase 2 alpha 1, DAPI 4',6-Diamidino-2-phenylindole dihydrochloride, FYN tyrosine-protein kinase Fyn, GAK cyclin G associated kinase, GST glutathione S-transferase, HEK-293T human embryonic kidney cell 293T, IP immunoprecipitation, LYN Lck/Yes-related novel protein tyrosine kinase, PCa prostate cancer, shRNA small hairpin RNA, SRPK1 serine/arginine-rich protein-specific kinase 1. **P < 0.01