Cyclotraxin b

Human dental pulp stem cells (DPSCs) were purchased from Lonza, Pharma, and Biotech (Cat. # PT-5025). The TrkB agonist (LM22A-4) was purchased from R&D System, the TrkB antagonist (Cyclotraxin-B, CTX-B) was purchased from Tocris Bioscience, and the recombinant human BDNF protein was purchased from Invitrogen. The MEMα, PBS, FBS, L-glutamine, and antibiotic–antimycotic were procured from Gibco™ Fisher Scientific (Waltham, MA, USA). Round, Poly-D-Lysine-coated (BioCoat™, 12 mm) German glass coverslips were purchased from Corning™ Fisher Scientific (Waltham, MA, USA). Various antibodies were procured: rabbit anti-BDNF (Santa Cruz, Dallas, TX, USA), rabbit anti-TrkB receptor (Proteintech, St. Louis, MO, USA), mouse anti-DMP-1 (R&D System/Sigma, St. Louis, MO, USA), and rabbit anti-DSPP (Santa Cruz, Dallas, TX, USA). Fluorescent secondary antibodies were obtained from Life Technologies (Grand Island, NY, USA). Lipoteichoic acid (LTA) from Staphylococcus aureus (Cat. # L2515) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

The desired concentration of each substance was achieved by the dilution of stock into the culture medium. Substances used were dicyclopropyl-linoleic acid (DCP-LA; Merck Life Science) 500 nM, selective PKCε activator; cyclotraxin B 10 nM (Tocris, Bio-Techne, Milan, Italy); and human recombinant BDNF 1 nM (Merk Life Since). Drug concentration was set in previous experiments (Kawano et al., 1997 (link); Bonalume et al., 2020 (link)), without toxic effects. Control cultures were treated with vehicle (DMSO). Differentiated SCs primary cultures were treated for the indicated times after overnight serum-free medium exposure. Medium changes and pharmacological treatments were done every other day.