Phosphate Uptake and Efflux Assays.

Monolayer cultures were seeded at 5 × 10⁵ cells per well in 1.5 mL medium in six-well plates; Pi transport assays were performed 24 h later at 37 °C. For Pi uptake assays, a previously published protocol was used (10) (i.e., the culture medium was replaced with phosphate-free DMEM [Gibco catalog number: 11971–025] plus 10% FBS). In early experiments, we labeled cells with 0.5 μCi/mL [³²P]-Pi (Perkin-Elmer; NEZ080001MC), but for health and safety reasons, we subsequently switched to 0.5 μCi/mL [³³P]-Pi (ARC; ARP 0153) without any impact on the data obtained. Thus, radiolabeled Pi is indicated as [*]Pi throughout this study. To assay [*]Pi uptake, cells were separated from medium by three rapid washes in 1 mL ice-cold inorganic phosphate-buffered saline (PBS). Cells were then lysed in 1 mL PBS plus 1% Triton X-100, and the accumulated [*]Pi was determined using a liquid scintillation counter. Uptake was normalized to cell protein as determined using a BCA protein assay (Pierce). For [*]Pi efflux assays, cells were loaded with [*]Pi for 20 min; then, cells were washed three times with 1 mL Pi-free DMEM and incubated with 1 mL DMEM supplemented with Pi (as an NaH₂PO₄/Na₂HPO₄ mixture, pH 7.4; 1 mM unless otherwise indicated). Aliquots (10% [vol/vol]) of medium were taken at the indicated times, and [*]Pi therein was determined. Experiments were terminated by cell lysis (see above), and residual cell-associated [*]Pi was determined; [*]Pi efflux is depicted as a percentage of total cellular [*]Pi at zero time.