C2C12 myoblasts produce uPA-dependent plasmin activity.

(A) C2C12 muscle cells express Plg. RT-PCR was performed to detect the expression of Plg in murine liver (positive control, lane 2) and differentiating C2C12 cells (lane 3). Lane 1 corresponds to PCR-negative control. Expected RT-PCR product size was 410 bp. (B) Plasmin activity is produced by C2C12 myoblasts. Histograms show plasmin specific activity (SA) (expressed as mOD/min/mg protein) in conditioned medium of C2C12 cells: preconfluent myoblasts (lane 1) and fusing myotubes (lane 2). No plasmin activity is detected in DMEM medium (lane 3), used as a negative control. The assays were performed in 0.1 M Tris-HCl and 2 mM EDTA, pH 7.6, containing the chromogenic substrate S-2251. (C) C2C12 cells produce uPA proteolytic activity. Conditioned media from preconfluent myoblasts (lane 1) and fusing myotubes (lane 2) cells were analyzed, after SDS-PAGE, for uPA and tPA activity, by PA zymography. The 45-kd approximate molecular mass species indicated by an arrow corresponds to murine uPA and was calculated according to standard molecular mass markers electrophoresed in an adjacent lane and stained with Coomassie blue. The photographs were taken after overnight incubation at 4°C and 4 hours at 37°C.