Investigating HDAC8 Regulation in A2058 Cells

A2058 cells were seeded at 1 × 10⁶ cells per plate in a 60 mm cell culture plate. After overnight incubation, the cells were treated with 0.1% DMSO or 20 µM PCI-34051 for 24 h. A2058 HDAC8 KO and OE cells were seeded at 1 × 10⁶ cells per plate in a 60 mm cell culture plate. Hypoxia was induced using 100 µM CoCl₂ for 6 h or by placing the cells in a hypoxia induction chamber. The total RNA from the cells was extracted using a FavorPrep^TM Blood/Cultured Cell Total RNA Mini Kit (Favorgen Biotech Corporation, Ping-Tung, Taiwan). One microgram of total RNA was reverse-transcribed into cDNA using an EasyScript™ cDNA synthesis kit (TransGen Biotech, Beijing, China). One microliter of cDNA was amplified using 10 µL of TOPreal™ qPCR 2X PreMIX SYBR green reagent (Enzynomics, Daejeon, Republic of Korea). A qPCR analysis was performed using the Applied Biosystems 7500 System (Applied Biosystems, Foster City, CA, USA). The mRNA values were normalized by GAPDH expression levels. The primers used are listed in Table 3.