Si nc

Human glioma cell lines (U251 and U87) and normal human astrocytes (NHAs) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, NY, USA) that was supplemented with 10% fetal bovine serum (FBS, Gibco), maintained at 37°C in a humidified incubator with 5% CO₂.
Small interfering RNAs (siRNAs) targeting ADAMTS9-AS1 (si-ADAMTS9-AS1#1: 5′-ACAGCTATATCAGCCAACCAGAGT-3′′ and si-ADAMTS9-AS1#2: 5′-ACCAGCCAGAGCAAGCTATATACT-3′), as well as a negative control (si-NC: 5′-GTTTACAACACGCTTCCTCTGA-3′) were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). U251 and U87 cells were plated in 6-well plates at a density of 1 × 105 cells per well and grown to reach 70-80% confluency, followed by a transfection with si-ADAMTS9-AS1#1, si-ADAMTS9-AS1#2, or si-NC for 48 h with lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA) complied with manufacturer’s instruction.

SELENOT gene silencing in H9c2 cardiomyocytes was performed as previously described by Rocca et al. (2022) [16 (link)]. Briefly, H9c2 cardiomyocytes (5000 per well) were seeded in 96-well plates and incubated for 48 h at 37 °C, 5% CO2. SELENOT siRNA (100 nmol/L) was transfected into H9c2 cardiomyocytes in serum-free medium using the Lipofectamine 2000 transfection reagent following the manufacturer’s instructions (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Negative control si-RNA (si-NC) was used to detect non-specific effects of siRNA delivery and to compare siRNA-treated samples. Both si-NC and siRNA for SELENOT were purchased from Santa Cruz Biotechnology. H9c2 cardiomyocytes were transfected in serum-free medium for 6 h, after which the medium was replaced with full-medium and cells were incubated for 36 h at 37 °C, 5% CO₂. H9c2 cells were treated with PA (100 µmol/L) or co-treated with PA and increasing concentrations of PSELT (from 5 to 100 nmol/L) for 24 h. At the end of the treatments, the viability of the H9c2 cells was evaluated by MTT assay. The cell viability was reported as the percentage cell survival relative to the si-NC transfected cells in six wells for each experimental group [16 (link)]. The experiment was repeated three independent times.