Fig. 2

ASPA overexpression inhibits PC-3 cell proliferation and migration in vitro. a Western blotting results for ASPA protein expression in PC-3 cells transfected with control or ASPA overexpression vector. Protein expression levels were normalized to β-actin levels. b CCK-8 assay showed that ASPA overexpression inhibited PC-3 cell proliferation. c Representative images of EdU-positive PC-3 cells transfected with control or ASPA overexpression vector (scale bar = 100 μm). The graph on the right shows the percentage of EdU-positive nuclei. The data were obtained from 7 fields of 3 independent experiments. d The colony formation assay showed that ASPA overexpression inhibited PC-3 colony formation. The graph on the right shows the colony numbers from 3 independent experiments. e RT-qPCR results of proliferation-related genes in PC-3 cells transfected with control or ASPA overexpression vectors. The mRNA expression levels were normalized to ACTB levels. f Western blotting results (left) and quantification results (right) for proliferation-related proteins in PC-3 cells transfected with control or ASPA overexpression vector. Protein expression levels were normalized to β-actin levels. g Transwell assays showed that ASPA overexpression inhibited PC-3 cell migration and invasion (scale bar = 100 μm). The graph on the right shows the migrating cells and the invading cells. The data were obtained from 8 fields of 3 independent experiments. h RT-qPCR results of epithelial–mesenchymal transition genes in PC-3 cells transfected with control or ASPA overexpression vector. The mRNA expression levels were normalized to ACTB levels. i Western blotting results (left) and quantification results (right) for epithelial–mesenchymal transition proteins in PC-3 cells transfected with control or ASPA overexpression vector. Protein expression levels were normalized to β-actin levels. j Flow chart of RNA-Seq in PC-3 cells transfected with control or ASPA overexpression vector. k Cluster analysis showed the global sample distribution profiles of the control group and ASPA overexpression group based on the RNA-Seq dataset in PC-3 cells. l GSEA results showed significantly altered cancer hallmarks based on the dataset of RNA-Seq in PC-3 cells from the control group and ASPA overexpression group. The data are presented as the mean ± standard deviation (SD). ASPA aspartoacylase, CCK-8 cell counting kit 8, CCND1 cyclin D1, CDH1 cadherin 1, CDH2 cadherin 2, EdU 5-ethynyl-2′-deoxyuridine, GSEA gene set enrichment analysis, MYC v-Myc myelocytomatosis viral oncogene homolog, MMP9 matrix metallopeptidase 9, NES normalized enrichment score, OD optical density, PCNA proliferating cell nuclear antigen, RNA-Seq RNA sequencing, RT-qPCR real-time quantitative PCR. **P < 0.01