Scintillation counter

A498 or UO-31 cells were plated in complete RPMI at 3,500 or 2,000 cells per well of a 96-well plate, respectively. After allowing cells to attach overnight, cells were treated with 0.1% DMSO (vehicle), or increasing concentrations of englerin A, C8-ceramide, or C8-ceramide-1-phosphate and incubated for 48 h. All conditions were conducted in triplicate. Cells were pulsed with [³H]-thymidine (1.6 μCi/well) during the last 7 h of incubation and then trypsinized and deposited onto glass microfiber filters using the Brandel M 24 cell harvester (Gaithersburg, MD). Incorporation of [³H]-thymidine into DNA was then determined by counting in a scintillation counter (Beckman Coulter, Fullerton, CA).

³H-Ouabain binding assay was performed as described previously [20 (link)]. The transport function of Na/K-ATPase was assessed by measuring the ouabain-sensitive uptake of the K⁺ congener, ⁸⁶Rb⁺, as described [20 (link)] with minor modifications. Briefly, cells were cultured in 12-well plates over 90% confluence and serum starved overnight before experiment. The cells were washed and incubated in culture medium with or without ouabain (1mM) over 10 minutes at 37°C. Monensin (20μM) was added to clamp extracellular Na⁺ to ensure maximal capacity of active uptake. ⁸⁶Rb⁺ (1μCi/well) as a tracer for K⁺, was then added for 10 minutes at 37°C and the reaction was stopped by washing three times with ice-cold 0.1M MgCl₂. Then cells were incubated in 10% trichloroacetic acid (TCA) and TCA soluble ⁸⁶Rb⁺ was counted in a Beckman scintillation counter. TCA-precipitated proteins were dissolved in 0.1N NaOH and 0.2% SDS solution and the concentration was determined using the BioRad Protein Assay Kit (BioRad Laboratories, Hercules. CA). All counts were normalized to protein amount.

Protein synthesis was measured as described by Gulve et al. [18 (link)] with the following modifications. L6 cells were plated in 48-well tissue culture plates and differentiated for 5 days. Cells were treated with 25 μM HMB for two hours in DMEM with 10% FBS and 0.8 mM L-Tyrosine and then for 1 h with 5 μM DEX in the absence or presence of effectors. Cells were then spiked with 1 μCi/ml of L-[ring-3,5–3H]-Tyrosine (Perkin Elmer, Waltham MA) and were incubated for 1 h. The reaction was stopped by placing the plates on ice. All wells were thoroughly washed twice with ice cold PBS media containing 2 mM non-radioactive L-Tyrosine (PBS-Tyr) and the cells then lysed in 0.1 ml of 0.1 mM NaOH/0.1% sodium deoxycholate. The cellular proteins were precipitated by adding 0.1 ml of cold 20% trichloroacetic acid (TCA; Sigma). This mixture was then incubated at 4°C for 15 min. After centrifugation (16,000×g for 10 min at 4°C), the pellet was washed once with cold 10% TCA and then, the precipitated proteins were dissolved in 0.1 ml of 1 M NaOH. An aliquot (5 μl) of the NaOH-solubilized material was used for total protein quantitation and the remaining dissolved proteins were neutralized with 1 M HCl, mixed with ReadySafe scintillation fluid (Beckman Coulter, Brea CA) and radiolabel determined with a scintillation counter (Beckman Coulter). Data was computed as dpm/μg of proteins.