Nnc 55 0396

The following antibodies and chemicals were used for this study: rabbit anti-α-tubulin (Abcam catalog #ab15246, RRID: AB_301787; 1:500), Rabbit anti-β3-tubulin (TUBB3; Covance catalog #MMS-435P, RRID: AB_2313773, 1:2000), mouse anti-MBP (BioLegend catalog #836504, RRID: AB_2616694; 1:1000), goat anti-Olig2 (R&D Systems catalog #AF2418, RRID: AB_2157554; 1:20), and mouse anti-O4 IgM (R&D Systems catalog #MAB 1326, RRID: AB_357617; 1:200). Alexa Fluor-conjugated secondary antibodies were all purchased from Thermo Fisher Scientific with a dilution of 1:500. nifedipine, verapamil, diltiazem, NNC 55-0396, ω-conotoxin GVIA (ω-CTX), ω-agatoxin IVA, SKF96365, cyclopiazonic acid (CPA), thapsigargin (Tg), and ryanodine (Ry) were all purchased from Sigma.

To determine cell survival and proliferation, cell growth was quantified using the CellTiter 96 AQ One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA). Cells were plated in 96-well culture plates at a density of 1–2 × 10⁴ cells/well in 100 μL of cell culture media. Cells were treated with different concentrations of mibefradil or NNC-55-0396 (Sigma-Aldrich, St. Louis, MO, USA). After drug exposure, 20 μL of CellTiter 96 AQ One Solution Reagent was added to each well and allowed to incubate for 2 h at 37 °C. The quantity of formazan product formed, which is directly proportional to the number of viable cells, was measured on a Multi-Mode Microplate Reader (MD SpectraMax M3, CA, USA) at 490 nm wavelength using a reference filter at 650 nm wavelength. Viability assays were performed at least three times in independent experiments, using triplicate measurements in each.