Fig. 1.

LacCer significantly upregulated [³H]-leucine incorporation in H9c2 cells: H9c2 cells were plated (10⁵ per well) in 24-well plates and allowed to proliferate in growth medium composed of DMEM supplemented with 10% fetal bovine serum. When cells had reached near confluence, growth medium was replaced with differentiation medium (DMEM containing 2% horse serum) for 48 h to induce differentiation of H9c2 myoblasts into myotubes. Cells were then stimulated for 48 h with a single dose of 100 µM PE and different glycolipids (as shown above, 100 µM each). [³H]-Leucine (5 Ci or 142 Ci/mmol) was included per well. At the end of the incubation period, cells were washed twice in PBS and proteins were subsequently precipitated with ice-cold 10% trichloroacetic acid. After dissolving the precipitates in 0.5 mol/L NaOH, 5 mL scintillation cocktail was added, and radioactivity was measured by liquid scintillation spectroscopy. [³H]-Leucine incorporation experiments were repeated five times with triplicate measurements for each experiment. LacCer upregulated protein synthesis in H9c2 cells significantly. *P < 0.003, **P < 0.004 vs control.