P2256

1 × 10⁵ MCF10A cells were seeded 24 hours prior to starvation in 35 mm glass-bottom dishes. Then, standard media was replaced with glucose-free, pyruvate-free, and serum-free DMEM (#11966, Gibco) overnight. FLIM images of NAD(P)H in three fields of view were taken in each dish immediately before and immediately after the replacement of media with fresh serum-free media containing either 10 mM sodium pyruvate or 10 mM sodium lactate (#P2256 and #L7022, Sigma). A total of 200–300 cells were imaged per condition, and individual cells were analyzed. This experiment was repeated in triplicate.

Human mesenchymal stem cells (MSC) were isolated from normal (non-diabetic) adult human bone marrow of normal volunteers and purchased from Lonza Cologne (Cologne, Germany). Incubation was performed as described [35 ]. The cells were maintained in 75 cm² flasks and cultivated in α-MEM (Cat. no. F0915; Biochrom, Berlin, Germany), supplemented with 20% FCS (fetal calf serum; Biochrom, Berlin, Germany) and 0.5 mg mL⁻¹ of gentamycin, 100 units mL⁻¹ penicillin, 100 mg mL⁻¹ of streptomycin and 1 mM pyruvate (#P2256 Sigma-Aldrich, Taufkirchen; Germany). Incubation was performed in a humidified incubator at 37 °C.
For cell viability and gene expression studies the MSC cultures were inoculated with 1 × 10⁴ cells per well (48 well plates (#CLS3548; Sigma-Corning, Taufkirchen, Germany)) in a total volume of 0.5 mL. The cultures were first incubated for a period of three days in the absence of the mineralization-activating cocktail (MAC). Subsequently, the cultures were used for the cell viability studies. For the gene expression studies the cells were incubated for a total period 21 days in the presence of MAC, comprising 50 mM ascorbic acid and 10 nM dexamethasone to induce biomineralization [36 (link)]. The third component usually used in the MAC, β-glycerophosphate, was omitted since polyP has been shown to be sufficient as a phosphate supply [37 (link)].

Primary neuronal cultures were prepared from the hippocampi of embryonic day 17.5 embryos from WT and PRRT2 KO mice of either sex, as previously described [12 (link)]. For low-density cultures, neurons were plated on poly-l-lysine (PLL; 0.2 mg/mL; Sigma #P2636)-coated 24 mm glass coverslips at a density of 120,000 cells per coverslip, and maintained as a sandwich co-culture with astroglia in modified Eagle’s medium (MEM Invitrogen, Carlsbad, CA, USA) supplemented with 1% N2 supplement (Gibco #1502-048), 2 mM l-glutamine, 1 mM sodium pyruvate (Sigma #P2256), and 4 mM glucose at 37 °C in 5% CO₂ humidified atmosphere [13 (link)]. When indicated, PLL-coated coverslips were subsequently coated with laminin (LN; 20 μg/mL; Sigma #L2020).

The kidneys were harvested from Ctns knockout mice and from their corresponding control littermates: one kidney was transversally split, and one half was fixed and processed for immunostaining while the other half was flash-frozen, homogenized by Dounce homogenizer in 1 mL of RIPA buffer that contains protease and phosphatase inhibitors and processed for western blot analysis. The contralateral kidney was taken to generate primary cultures of mPTCs^{4 (link)}. Freshly microdissected PT segments were seeded onto collagen-coated chamber slides (C7182, Sigma-Aldrich) and/or collagen coated 6- or 24-well plates (145380 or 142475, Thermo Fisher Scientific), and cultured at 37 °C and 5% CO₂ in DMEM/F12 (21041-025, Thermo Fisher Scientific) with 0.5% dialyzed fetal bovine serum (FBS), 15 mM HEPES (H0887, Sigma-Aldrich), 0.55 mM sodium pyruvate (P2256, Sigma Aldrich), 0.1 ml L⁻¹ non-essential amino acids (M7145, Sigma Aldrich), hydrocortisone, human EGF, epinephrine, insulin, triiodothyronine, TF, and gentamicin/amphotericin (Single Quots® kit, CC‒4127, Lonza), pH 7.40, 325 mOsm kg⁻¹. The medium was replaced every 48 h. Confluent monolayers of mPTCs were expanded from the tubular fragments after 6–7 days, characterized by a high endocytic uptake capacity. These cells were negatively tested for mycoplasma contamination.

HepG2 TMEM127 KO or control cells were transferred to a 12-well plate at a density of 1 × 10⁶ cells/well and cultured for 28 h incubated in glucose-free Dulbecco’s modified Eagle’s medium (Thermo Fisher Gibco, catalog no. D1050) supplemented with 2 mm-pyruvate(Sigma, catalog no. P2256) or phosphate-buffered saline (PBS) overnight, then Insulin 100 μm or PBS were added for 30 min and cells were harvested for RNA.