Investigation of public genomic databases
The institutional review board of Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC approved this study (TSGHIRB No: B-111-21). Several researchers have demonstrated the process for investigating public genomic databases [12-14]. For the purpose of exploring the impact of DPY19L1 expression on pathological grading and overall survival, we conducted analysis of The Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA) . The data from TCGA database was extracted through UCSC Xena (https://xenabrowser.net/) and GlioVis (http://gliovis.bioinfo.cnio.es/) [16, 17], consisting of 226 sheets grade II gliomas, 244 sheets grade III gliomas and 150 sheets grade IV gliomas. We additionally acquired 321 samples from the CGGA database (http://www.cgga.org.cn/) including 103 grade II gliomas, 79 grade II gliomas and 139 grade IV gliomas.
We used Kaplan-Meier survival curve to present survival outcome compared to different expression level from cohorts of TCGA and CGGA. According to statistical analysis, the cut-off point of DPY19L1 expression was considered as median value. Plots were produced by GraphPad Prism 5 software, and P < 0.05 was defined as statistical significance.
RNA isolation and quantitative real-time PCR
We extracted total mRNA via TRIzol™ Reagent (Thermo Fisher Scientific, Wal-tham, WA, USA) according to the manufacturer’s protocol. Oligo dT primer with MMLV Reverse Transcriptase (Epicentre Biotechnologies, Madison, WI, USA) was applied for cDNA synthesis. We purchased normal brain cDNA from Origene Technologies (Rockville, MD, USA). Amplification and quantification of DPY19L1 expression were achieved by a StepOne™ Real-Time PCR System (Thermo Fisher Scientific, USA). We used the 2−ΔΔCt method to compare relative quantitative gene expression with GAPDH as an internal control. The primer pairs included presented below: DPY19L1 forward 5′- ACACCACCTCTCCGTGAAAGCT-3’ and reverse 5′- GCAGAGTGCAATCAA-GCTTCCTC-3’; GADPH forward 5′-CTTCATTGACCTCAACTAC-3′ and reverse 5′-GCCATCCACAGTCTTCTG-3′.
LN229, U118MG and U87MG cell lines were commercially available from American Type Culture Collection (ATCC), and we also purchased GBM8401 glioma cell line from Bioresource Collection and Research Center (BCRC number 60163, Hsinchu, Taiwan). LN229 and GBM8401 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) which comprises 2% fetal bovine serum (FBS), penicillin, and streptomycin, and U87MG, U118MG, and LNZ308 cells were cultured in DMEM consisting of 10% FBS, penicillin, and streptomycin. Cells above were incubated in 37 °C and 5% CO2 condition.
Cell Lysate Preparation and Western Blot
RIPA buffer (100 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, and 1% Triton-X-100) was applied for cell lysis at 4 °C for 10 min and centrifuged at 15,000 rpm for 10 min. Separation of proteins was achieved by 10% Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then we transferred the proteins to polyvinyl difluoride membranes (Millipore, MA, USA) which was blocked with 5% skim milk in TBST for 1 hour. DPY19L1 antibody (Atlas Antibodies, Stockholm, Sweden) and GADPH (Santa Cruz Biotechnology, Inc.) were incubated. The result was observed through enhanced chemiluminescence and ChemiDocTM MP Imaging System (BIO-RAD, Hercules, CA, USA).
Immunohistochemical Staining of Glioma Tissue Microarray
We acquired samples of high- and low-grade gliomas from the Human Protein Atlas (https://www.proteinatlas.org/ENSG00000173852-DPY19L1/pathology/glioma). 11 cases with 19 cores of tissue microarray were stained using DPY19L1 antibody (HPA059139) and immunohistochemical staining protocol.
Protein–protein network and signaling pathways analysis
To analyze interaction between DPY19L1 and associated proteins, we investigated the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database version 11.5 (http://string-db.org) .
We performed single tail t test to clarify the significance of DPY19L1 expression in different glioma grades according to the databases (GDS1962 / 1560916_a_at, TCGA and CGGA). The Bonferroni method was conducted to accurate the P value and eliminate the possibility of type I error by R 3.0.1 software package.