Figure 3. A quantitative cell detachment assay was used to investigate the involvement of myosin contractility in differentiated L6 cells.

The amount of detached cells is expressed as % of basal where basal means cells that are not treated with any drugs before exposure to trypsin·EDTA for 20 min. A) Cells were either kept in culture media (CM) during the whole experiment or expored to Trypsin·EDTA (T-E) for 20 min. T-E+ISO indicated cells pre-treated for 30 min of 1 µM isoprenaline (ISO) before the 20 min exposure to T-E in the continuous presence of isoprenaline (***). Histograms show percentage of unstimulated cells detached by T-EDTA expressed as mean ±SEM of four independent experiments. ** p<0.01, *** p<0.001, one-way ANOVA versus control cells in T-EDTA. B) L6 myotubes kept in Trypsin·EDTA with (triangels) or without (squares) isoprenline treatment. Aliquots of the detached cells were harvested and counted at the timepoints 10, 15, 20 and 25 min. C) L6 myoutbes kept in trypsin alone with (triangles) or without (squares) isoprenaline treatment. Aliquots of the detached cells were harvested and counted at the timepoints 10, 15, 20, 25 and 30 min. D) L6 myotubes were kept in Trypsin·EDTA for 20 min after 0 or 30 min pretreatment with isoprenaline. In both C and D, point show % of cells detached after 20 min in Trypsin·EDTA. *** p<0.001 with one-way ANOVA for iso and acute iso versus control. ** p<0.01 for iso versus acute iso. E) L6 myoblasts were kept in Trypsin·EDTA for 3 min after 0 or 30 min pretreatment with isoprenaline. *** p<0.001 with one-way ANOVA for iso and acute iso versus control. ** p<0.01 for iso versus acute iso. F) 30 min pre-treatment of 50 µM of the myosin-inhibitor blebbistatin (blb) or 30 min of 1 µM ISO pre-treatment followed by 20 min of T-E exposure. *** p<0.001, one-way ANOVA versus T-EDTA.