Hbepc

Lung adenocarcinoma cell lines (NCI-H23, HCC827, SPC-A1, and A549) and human bronchial epithelial cells (HBEpC) were all obtained from ATCC. They were cultured in RPMI 1640 culture medium containing 10% fetal bovine serum in an incubator at 37°C and 5% CO₂. The oligonucleotides including siRNAs (si-SOX2: 5′-GGAGCACCCGGAUUAUAAA-3′; si-Myc: 5′-CAUGGUGAACCAGAGUUUC-3′; si-p53: 5′-UCU ACAAGCAGUCACAGCA-3′; si-STAT3: 5′-GGAGGCAUUC GGAAAGUAU-3′), ASO-ST8SIA6-AS1 (5′-GGGUUUGUG CAAGCAAACU-3′), and miR-125a-3p mimics/inhibitors were designed and commercially purchased from RiboBio (Guangzhou, China). p53-overexpressing pcDNA 3.0 vector was obtained from Thermo Fisher (MA, United States). Transfection was performed using Lipofectamine 3000 (Thermo Fisher) with 10 nM final concentration as per standard protocol, and the transfection efficiency was detected after 48 h using real-time quantitative PCR (qRT-PCR) analysis.

The cell lines U87, and A549 were obtained from ATCC, HBEpC (Human Bronchial Epithelial Cells) was obtained from Cell Applications (San Deigo, California) and MDA-231-BR was kindly donated by Patricia Steeg, NIH/NCI. The U251MG glioblastoma cell line (formerly known as U-373 MG) was originally obtained from ATCC (Manassas, VA) and was used for a maximum of fifteen passages. U87, U251MG, A549 and MDA231-BR cells were maintained in DMEM (Gibco) supplemented with 2 mM L-glutamine (Gibco) and 10% fetal bovine serum (Gibco). HBEpC were grown in bronchial/tracheal epithelial growth medium obtained from Cell Applications and were used for a maximum of three passages. All cells were maintained at 37°C and 5% CO₂. The cells were seeded into 384 well plates and allowed to grow for 24hrs and then treated as specified. After a 72 hr exposure to the indicated compounds the cells were stained with Hoescht 33342 and ethidium homodimer I for total and dead cell counts, respectively. Cells were imaged using an In Cell Analyzer 2200 and cell viability was measured based on viable nuclei count. For these studies DDC was dissolved in sterile water, DSF and Cu(DDC)₂ were solubilised in DMSO and cells were dosed such that the final DMSO concentration was 0.05%.