Bovine serum

The immortalized human foetal osteoblast hFOB 1.19 cell line (ATCC, CRL-11372) was used to generate the TMEM38B knock out model. Cells were grown at 34°C in humidified atmosphere containing 5% CO₂ in growing medium made of Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM:F12) (Sigma Aldrich, St. Louis, Missouri, USA) containing 2.5 mM L-glutamine and 15 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES) and added with 10% bovine serum (Euroclone, Buchs, Switzerland) and 0.3 mg/ml geneticin (G418).
For the differentiation and mineralization experiments, cells were kept in DMEM media (Lonza Biosciences, Basel, Switzerland) supplemented with 100 nM dexamethasone (Sigma Aldrich), 50 μg/ml ascorbic acid 2-phosphate (Fluka) and 10 mM β-glycerophosphate (Sigma Aldrich). Cells were maintained in this medium at 37° C for 4, 8, 15 or 21 days depending on the experiments.
For each experiment the not transfected human foetal osteoblasts were used as control.

SH-SY5Y human neuroblastoma cells (Sigma-Aldrich, St. Louis, MO, USA, from The European Collection of Authenticated Cell Cultures, ECACC, Public Health England) were used. This cell line is not listed as a commonly misidentified cell line by the International Cell Line Authentication Committee (ICLAC; http://iclac.org/databases/cross-contaminations/, on 16 January 2023) and was not further authenticated during the last five years. The SH-SY5Y cells were grown in Dulbecco’s Modified Eagle Medium plus F12 in a 1:1 ratio, containing 10% bovine serum, 1% L-glutamine, and 1% penicillin–streptomycin (all from Euroclone, Italy); they were maintained at 37 °C in a humidified 5% CO₂ atmosphere and used within passage 30. For the Western blot (WB) and Co-IP experiments, the cells were seeded in six-well plates at a density of 500,000/4 mL/well. Under these conditions, the cells are not differentiated into neurons. Twenty-four hours after the onset of the culture, the cells were treated for 5 min with CGS 21680 (300 nM) or DHPG (10 µM). ZM 241385 (500 nM) and MPEP (10 µM) were applied 20 min before and then along with CGS 21680 or DHPG. The SH-SY5Y cells were maintained at 37 °C under 5% CO₂ for the duration of the experiments.