Supplementary MaterialsAdditional file 1: Table S1. to determine the effect of ACYP2 knockdown around the expression of NF-kBs downstream targets (Bcl-xL and Bcl-2) in the indicated cells. Physique S9. qRT-PCR was used to determine the effect of ACYP2 knockdown around the expression of c-Mycs downstream targets (E2F2 and cyclin E) and p-STAT3s downstream targets (c-Fos and c-Jun). Physique S10. Cells transfected NVP-BSK805 dihydrochloride with the indicated constructs were treated with the vehicle or Stattic, and the MTT assay was then carried out to evaluate their effect on cell proliferation. Physique S11. qRT-PCR assay was performed to determine mRNA expression levels of PMCA1C4 in the indicated glioma cell lines. 13046_2020_1607_MOESM2_ESM.docx (17M) GUID:?5F9531B6-33F8-4E34-A987-DC33F7CA7732 Data Availability StatementAll data generated or analyzed during this study are included in this article. Abstract Background Acylphosphatase 2 (ACYP2) is usually involved in cell differentiation, energy metabolism and hydrolysis of intracellular ion pump. It has been reported as a negative regulator in leukemia and a positive regulator in colon cancer, respectively. However, its biological role in glioma remains totally unclear. Methods We performed quantitative RT-PCR (qRT-PCR), immunohistochemistry (IHC) and western blot assays to evaluate ACYP2 expression. The NVP-BSK805 dihydrochloride functions of ACYP2 in glioma cells were determined by a series of in vitro and in vivo experiments, including cell proliferation, colony formation, cell cycle, apoptosis, migration, invasion and nude mouse tumorigenicity assays. In addition, western blot and co-immunoprecipitation (Co-IP) assays were used to identify its downstream targets. Results Knocking down ACYP2 in glioma cells significantly inhibited cell proliferation, colony formation, migration, invasion and tumorigenic potential in nude mice, and induced cell cycle arrest and apoptosis. Conversely, ectopic expression of ACYP2 in glioma cells dramatically promoted malignant NVP-BSK805 dihydrochloride phenotypes of glioma cells. Mechanistically, ACYP2 promoted malignant progression of glioma cells through regulating intracellular Ca2+ homeostasis via its conversation with PMCA4, thereby activating c-Myc and PTP1B/STAT3 signals. This could be effectively reversed by Ca2+ chelator BAPTA-AM or calpain inhibitor calpeptin. Conclusions Our data demonstrate that ACYP2 functions as an oncogene in glioma through activating c-Myc and STAT3 signals via the regulation of intracellular Ca2+ homeostasis, and indicate that Rabbit Polyclonal to ASC ACYP2 may be a potential therapeutic target and prognostic biomarker in gliomas. and rRNA, and each sample was run in triplicate. The primer sequences were summarized in Additional?file?1: Table S1. Cell lines and drug treatments Human glioma cell lines U251, SHG44, A172, U87, BT325 and SF295 were provided by Cell Lender of the Zhongshan University or college. A172 and BT325 was provided by Kunming Cell Lender of The Chinese Academy of Sciences. Cells were all routinely cultured at 37?C in DMEM medium with 10% fetal bovine serum (FBS). All cell lines used in this study were authenticated by short tandem repeat (STR) analysis in Genesky Co. Ltd. (Additional file 1: Table S2), and the results was completely consistent with previous studies  and database (Cellosaurus: https://web.expasy.org/cellosaurus/). In some experiments, cells were treated with 100?M cell-permeable c-Myc-Max dimerization inhibitor 10,058-F4 (Selleck Chemicals) for 48?h to inhibit transcriptional activity of c-Myc. Cells were treated with 5?M BAPTA-AM (Selleck Chemicals) for 6?h to chelate intracellular Ca2+. Cells were treated with 10?M calpeptin (Selleck Chemicals) for 12?h to block calpain activity. Cells were treated with 10?M sodium orthovanadate (Na3VO4) for 1?h to inhibit PTP1B activity. The same volume of the vehicle was used as the control. siRNAs, expression plasmids and lentivirus transfection Oligonucleotides of siRNAs targeting ACYP2, PMCA4 and PTP1B were obtained from Gene Pharma (Shanghai, China) and Ribobio (Guangzhou, China), respectively. The sequences were presented in Additional file 1: Table S3. Cells were transfected at 50% confluence using Lipofectamine 2000 (Invitrogen, Grand Island, NY) according to the instructions of the manufacturer, with a final siRNA concentration of 50?nM. All silencing experiments were carried out in triplicate. Two oligonucleotides.