Fig. 5

ASPA negatively regulates JNK1/2-C-Jun activity. a Luciferase activity of 45 pathways affected by ASPA overexpression or knockdown. Blue and red indicate down- and up-regulated activity, respectively. Dot and triangle indicate ASPA versus control and shASPA versus shRNA, respectively. b Enrichment of the AP-1 pathway in the control group and ASPA overexpression group and analysis by GSEA based on RNA-Seq dataset in PC-3 cells. c Heatmap showed the significantly altered genes related to the AP-1 pathway based on the RNA-Seq dataset in PC-3 cells transfected with the control or ASPA overexpression vector. d Western blotting results (left) and quantification (right) results for ASPA and phosphorylation of AP-1 (C-Jun and C-Fos) in PC-3 cells transfected with control or ASPA overexpression vectors. Protein expression levels were normalized to β-actin levels. e Western blotting results (left) and quantification (right) results for ASPA and phosphorylation of AP-1 (C-Jun and C-Fos) in PC-3 cells transfected with shRNA or shASPA. Protein expression levels were normalized to β-actin levels. f Western blotting results for ASPA and phosphorylation of C-Jun in PC-3 cells transfected with control, ASPA overexpression vector, or C-Jun overexpression vector. Protein expression levels were normalized to β-actin levels. g Cell proliferation ability of PC-3 cells cotransfected with ASPA overexpression vector and/or C-Jun overexpression vector was assessed using a CCK-8 assay. h The cell migration ability of PC-3 cells cotransfected with ASPA overexpression vector and/or C-Jun overexpression vector was assessed using a Transwell assay. The graph on the right shows the migration of cells in the treatment group relative to the control group. The data were obtained from 8 fields of 3 independent experiments (scale bar = 100 μm). i Western blotting results of JNK1/2, P38, and ERK1/2 phosphorylation in PC-3 cells transfected with control or ASPA overexpression vector and transfected with shRNA or shASPA. Protein expression levels were normalized to β-actin levels. j Western blotting analysis showed the expression of PCNA and cyclin D1 and the activity of JNK1/2 and C-Jun in PC-3 cells transfected with shASPA and/or treated with the JNK inhibitor JNK-IN-8. k The proliferation ability of PC-3 cells transfected with shASPA and/or treated with JNK-IN-8 was assessed using a CCK-8 assay. The data are presented as the mean ± standard deviation (SD). ASPA aspartoacylase, AP-1 activator protein-1, CCK-8 cell counting kit 8, C-Jun v-Jun avian sarcoma virus 17 oncogene homolog, C-Fos v-Fos FBJ murine osteosarcoma viral oncogene homolog, DMSO dimethyl sulfoxide, ERK extracellular regulated protein kinases, GSEA gene set enrichment analysis, FDR false discovery rate, JNK c-Jun N-terminal kinase, NES normalized enrichment score, OD optical density, PCNA proliferating cell nuclear antigen, PID Pathway Interaction Database, RT-qPCR real-time quantitative PCR, shRNA small hairpin RNA. **P < 0.01, ns not significant