HDAC6 Knockdown in Human Dental Pulp Cells

For functional knock-down of HDAC6, an HDAC6-targeting small interfering RNA (siRNA; siHDAC6, Sigma) and the universal negative control siRNA were transfected into hDPCs. Cells were seeded into 6-well plates and then transfected with siRNA using Lipo2000 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer's instructions (Tao et al., 2019 (link)). Knockdown efficacy was confirmed by western blot. Odontoblastic differentiation was induced 48 h after transfection. For the tracing of autophagic flux, mRFP-GFP-LC3 plasmid (plasmid 21074; Addgene) was transfected into hDPCs following transfection with siRNA prior to the induction of odontoblastic differentiation using Lipo2000 according to the manufacturer's instructions(Kimura et al., 2007 (link)).