Poly dlysine coated

For the overexpression experiments, pCMV6 entry vectors containing the open reading frames (ORF) of the five candidate mouse proteins mOCIAD1, mPAFAH2, mHTATIP2, mPDCD6 and mSAR1B were purchased from Origene Technologies GmbH (Herford, Germany). To equip the proteins with C-terminal myc-tags, the ORFs were amplified by RT-PCR, using gene-specific primers equipped with appropriate restriction cites (see Table S3), and recloned into pCMV3A mammalian expression vectors (Agilent Technologies, Santa Clara, CA, USA). The correct insertion and preservation of the ORF was subsequently checked by sequencing (Eurofins Genomics, Ebersberg, Germany).
For the colocalization experiments, HepG2 cells and mouse embryonic fibroblast (for preparation see [18 (link)]) were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS, 1% penicillin/streptomycin. For immunofluorescence microscopy, the cells were seeded into 24-well cell culture plates (Sarstedt, Nümbrecht, Germany) equipped with Poly-D-lysine-coated (Thermo Fisher Scientific, Waltham, MA, USA) glass coverslips. For overexpression experiments, the cells were transfected on the next day with LipofectamineTM3000 reagent (Thermo Fisher) containing 0.5 μg plasmid DNA per well. After 24 h of incubation, the cells were fixed with 4% paraformaldehyde (PFA) in PBS (pH 7.4) for 20 min, at RT.

Immediately following loss of the tooth, it was placed in media (DMEM/F12 50/50 mix with HEPES) (Thermo Fisher Scientific), 100 U/mL penicillin (Thermo Fisher Scientific), and 100 μg/mL streptomycin (Thermo Fisher Scientific). DPSCs were isolated and cultured as previously described (41 (link)). Briefly, after mincing the dental pulp from inside the tooth cavity, 3 mg/mL Dispase II (Thermo Fisher Scientific) and 4 mg/mL Collagenase I (Thermo Fisher Scientific) were added to digest the tissue. Cells were then seeded on poly-d-lysine–coated (Thermo Fisher Scientific) 12-well plates with DMEM/F12 1:1, 10% (v/v) fetal bovine serum (Thermo Fisher Scientific), 10% (v/v) newborn calf serum (Thermo Fisher Scientific), 100 U/mL penicillin, and 100 μg/mL streptomycin. Then, 80% confluent cultures were passaged with Tryple E express (Gibco, Thermo Fisher Scientific) and neuronal differentiation performed only on early-passage cells (<4).