Gene Expression Analysis of U251 Cells

As described in our previous study [23 (link)], a gene expression study was performed using qRT-PCR. A complete list of primers is provided in Table 1. U251 cells were seeded in a 6-well plate at a density of 1 × 10⁵ cells/well and cultured in DMEM + 10% (v/v) FBS until their confluence reached 90%. Once the target confluence was reached, the medium was replaced with fresh 75% (v/v) hMSC-CMs in DMEM + 10% (v/v) FBS and the cells were cultured for another 72 h. U251 cells cultured in 75% (v/v) U251-CMs in DMEM + 10% (v/v) FBS served as controls. At the end of the culture, total RNA was isolated from treated U251 cells using TRIzol™ reagent (Sigma-Aldrich, U.S.A.), according to the manufacturer’s instructions. The isolated RNAs were then used to determine the expression levels of target genes by iTaq Universal One-Step RT-qPCR Kits (Bio-rad, U.S.A.), according to the manufacturer’s instruction. PCR was carried out using an Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystem, U.S.A.) with the following setting: initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation (95°C, 10 s), annealing (60°C, 10 s), and extension (72°C, 40 s). The mRNA level of each target gene was normalized with the mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using a 7500 software version 2.0.5 (Applied Biosystem, U.S.A.).