Vitamin a

Neuroblastoma cells were seeded in 96-well plates at a density of 10³ cells/well in a defined serum-free medium: DMEM/F12 based medium (as detailed in Cell lines) w/o FCS, supplemented with 10 ng/mL EGF (Sigma-Aldrich), 20 ng/mL FGF2 (Sigma-Aldrich), and 1× B-27 supplement w/o vitamin A (Gibco). After 24 h, cells were treated by the addition of fresh medium with the selective HTR3A receptor agonists, N-methylquipazine dimaleate (NMQ; Tocris, cat. #0566) or SR57227 (Tocris, cat. #1205), or the HTR3A receptor antagonists, granisetron hydrochloride (Tocris, cat. #2903) and VUF 10166 (Tocris, cat. #10166). In case of HTR3A receptor antagonists, medium was further supplemented with 5HT (Merck, cat. #14927) to the final concentration of 1 µM to evaluate the effect of HTR3A receptor inhibition. The proliferation activity was analyzed after additional 5 days using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) at a final concentration of 455 μg/ml^{100 (link)}. The medium with MTT was replaced by 200 μL of DMSO per well after a 3 h incubation under standard conditions in order to solubilize the MTT product. The absorbance was measured at 570 nm with a reference absorbance at 620 nm wavelength using a Sunrise Absorbance Reader (Tecan).

Kcnk9KO^mat primary cortical neurons (mPCNs) were isolated from E14 embryos derived from matings between female Kcnk9KO^hom mice and WT males. After collection of brains, cortices were dissected out and mechanically separated into single cortical cells through resuspension. The mPCNs were plated on Poly-L-Ornithine (Sigma)- and Laminin (Sigma)-coated plates and maintained in culture medium containing Neurobasal medium (Gibco), supplemented with 2% B27 plus vitamin A (Gibco) and 1% Glutamax (Gibco), in a humidified incubator at 37 °C and 5% CO₂.