Myo-[2-3H]inositol cell labeling and HPLC

Cells were labeled with myo-[2-³H]inositol following our previous protocols [19, 37, 41], Briefly, cells (in 35 mm dishes) were maintained for 24 h in “starvation” medium (glucose- and inositol-free DMEM, containing 10% dialyzed FBS, 5 μg/ml each of insulin and transferrin, 2 mM pyruvate, 25 mM HEPES (pH 7.4), 50 units/ml penicillin, and 50 μg/ml streptomycin), in a 37°C incubator under a humidified, 5% CO₂ atmosphere. The medium was then replaced with fresh starvation medium supplemented with 25 μCi/ml myo-[2-³H]inositol (corresponding to ~1.5 μM final concentration of myo-inositol) and cells were labeled for 25 h. Cell treatments were performed in the same labeling medium for 1–2 additional hours as specified in the figure legends. The incubation time was sufficient to reach a condition of 90–100% isotopic equilibrium (i.e., steady-state) across the lipid pools as calculated from the PIP/PI ratios in both HEK293 cells and podocytes as described previously [37, 40, 42]. Cells were washed 3 times in PBS carrying phosphatase inhibitors (50 mM NaF, 10 mM Na pyrophosphate, 25 mM Na β-glycerophosphate, and 2 mM Na metavanadate) and lipids were extracted with TBAS reagent [CH₃OH/1M HCl (1:1 v:v)] in the presence of 5 mM EDTA and 5 mM tetrabutylammonium sulfate], deacylated at 54°C for 60 min with methylamine reagent (6.4 ml methanol, 3 ml 40% w/w methylamine, 1.3 ml water, 1.1 ml n-butanol, with 1 mM Na EDTA, pH 8). Glycerophosphoinositol phosphates (GroPInsP) were analyzed by HPLC (Waters 5215) on a 5-micron Partisphere SAX column eluted as previously described [19, 37, 39, 41, 43]. Fractions were collected every 0.25 min and analyzed for [³H] radioactivity after the addition of scintillation mixture. Data evaluation and documentation was performed by Microsoft Excel. Individual peak radioactivity was quantified by area integration and presented as a percentage of the summed radioactivity from the [³H]GroPIns3P, -4P, -5P, -(3,5)P₂, and -(4,5)P₂ peaks (“total radioactivity”).