Fig. 3. A soluble factor is secreted by myotubes in response to IGF-1 treatment and recruits reserve cells for fusion. (A) The ability of conditioned medium from total cultures (myotubes + reserve cells) or reserve cell cultures (obtained after myotube removal by brief trypsinization) treated with IGF-1 to induce an increase in the fusion index was measured. (B) The ability of neutralizing antibodies directed against IL-13, IL-4, IL-15 to prevent the increase in cell fusion induced by IGF-1 was tested. Mouse immunoglobulins were used as a control. Above each column the significance of the sample versus the control (upper) and versus the IGF-treated sample (lower) is given; NS, non significant; ^**P<0.01 and ^***P<0.001. (C) The effects of IGF-1 on the expression of IL-13, IL-4 and IL-15 was investigated in total cultures, by RT-PCR analysis, and normalized with GAPDH.