Dulbecco s modified

Sulforaphane (SFN) (Cat #S4441, Sigma-Aldrich, St. Louis, MO, USA) and Brusatol (BRU) (Cat #SML1868, Sigma-Aldrich) were solubilized in DMSO and used to treat cells based on our prior findings [12 (link)]. All treatments were prepared in traditional cell culture growth media composed of high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) with sodium pyruvate (Cat #25-500, Genesee Scientific, San Diego, CA, USA) and 10% fetal bovine serum (Cat #F4135, Genesee Scientific). Before treatment, MACT cells were seeded in 6-well plates at a seeding density of 3 × 10⁵ cells per well in DMEM media and grown overnight in an incubator at 37℃/5%CO₂. Three replicates of MACT cells were then treated with either 10 µM SFN, 100 nM BRU, or an equivalent amount of DMSO (i.e., control) for 24 h before the isolation of RNA. Cells were cultured, and treatments were applied in 2 mL of media/well.

P19 cells were differentiated into neurons over a four-day-long process as described previously [29 (link)]. In summary, cells were seeded at a density of 1 × 10⁶ cells per dish in a differentiation medium comprised of Dulbecco’s Modified Eagle’s Medium (DMEM; Genesee Scientific) supplemented with FBS (5% v/v), antibiotics, and retinoic acid (1 μM; Sigma-Aldrich, St. Louis, MO, USA) to promote neurogenesis. Cells were cultured on a rotating stage to promote aggregate generation [30 (link)]. The medium was changed on day two of differentiation. On day four, aggregates were plated on cell culture plates in a post-differentiation maintenance medium comprised of DMEM supplemented with FBS (10% v/v) and antibiotics. Differentiated cells were cultured for two additional days before being used for subsequent experiments.