Shrna

Knockdown of AK139328 expression was performed using short hairpin RNA (shRNA) targeting AK139328 (shRNA-AK139328-1; 5′-CACCGGAAACTCAGCTATCACATGCCGAAGCATGTGATAGCTGAGTTTCC-3′), shRNA-AK139328-2 (5′-CACCGCAGCAGAAAGACATGTTTGGCGAACCAAACATGTCTTTCTGCTGC-3′) and corresponding scrambled negative control (shRNA; 5′-CACCTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTTG-3′), which were both synthesized by Shanghai GenePharma Co., Ltd. PC12 cells were transfected with 500 ng/µl shRNA-AK139328-1/2 or shRNA using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The cells were harvested for use in subsequent experiments at 48 h post-transfection.

Cell transfections were performed using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Untransfected cells were used as a blank control. Briefly, 4×10⁵ H2122 or A549 cells were plated into a six-well plate and cultured to 90–95% confluence. The overexpression of GSTO1 in NSCLC cells was induced through transfection with a pcDNA3.1 plasmid (4 µg; Invitrogen; Thermo Fisher Scientific, Inc.) carrying the GSTO1 cDNA insert, with an empty vector (4 µg) as the negative control (pcDNA3.1-NC). The knockdown of GSTO1 in NSCLC cells was induced using 2 µg short hairpin RNA (shRNA; Thermo Fisher Scientific, Inc.) against GSTO1, using stable non-specific shRNA (2 µg) as the NC (shRNA-NC). The shRNA-NC sequence was 5′-CCGGCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGGTTTTTC-3′ and the shRNA-GSTO1 sequence was 5′-GCTGGAAGCAATGAAGTTA-3′. Cells were transfected for 24 h at 37°C in an atmosphere containing 5% CO₂ to obtain stably transfected cells for future use. At 48 h post-transfection, the overexpression and knockdown of GSTO1 was confirmed using reverse transcription-quantitative PCR (RT-qPCR) and western blotting.