Pre 084

HTM5 cells were plated in serum-free fresh medium in 96-well plates (Sarstedt) at a density of 40 000 cells/well (n = 6 wells/group) and treated with 20 ng/mL of PDGF, 5–15 µM FLU (PDGF + FLU), 5–15 µM SA-4503 (PDGF + SA; SA−4503: 165377-44-6 Tocris Bioscience, Bristol, UK) and 5–15 µM PRE-084 (PDGF + PRE; PRE−084: P2607 Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Control cells were treated with vehicle (HCl) alone. After the treatment period, cells were incubated with methyl-thiazoletetrazolium (MTT, M6494 Invitrogen, Carlsbad, CA, USA) for 4 h followed by solubilization in DMSO-ethanol (1:1). The formation of water-insoluble formazan was determined by measuring optical density at 570 nm in a Plate CHAMELEON™ V Fluorometer-Luminometer-Photom reader (Hidex, Turku, Finland). Lactate dehydrogenase (LDH) assay (C20300, Invitrogen, Carlsbad, CA, USA) was performed from the supernatant of the cells according to the manufacturer’s protocol, and samples were measured in the same way as in case of MTT.

siRNA against Sig1R was obtained from Life Technologies. A mitochondrial Case12‐expressing plasmid (pCase12‐mito) was purchased from Evrogen (Moscow, Russia). N2a‐IP₃R3 cells were seeded at 2.0 × 10⁵ cells/dish on poly‐D‐lysine‐coated glass‐bottom dishes (MatTek, Ashford, MA). After transfection with Lipofectamine 2000 and/or Lipofectamine RNAi MAX (Life Technologies), the cells were incubated for 24 h in the differentiation media. Fluo‐4 AM (Life Technologies), PRE‐084, or NE‐100 (both from Sigma‐Aldrich, 5 μM each) was added 1 h prior to observation. The cells were washed twice with warmed Hank's balanced saline (Life Technologies). Fluorescence intensity was measured every 3 s until 120 s, and intracellular Ca²⁺ release was stimulated by adding adenosine triphosphate (ATP; 10 μM) to the media at 30 s.