Fig. 4

MMP3 secreted from TMSC promotes collagen IV degradation and BMC migration in vitro. a Immunohistochemical analyses of bone marrow basement membrane from mice sacrificed on day 10. Slides were stained with anti-collagen IV antibody and counterstained with hematoxylin. Representative images with a higher magnification focusing on bone marrow endothelium in lower panel. Scale bar 50 μm. b The enzymatic activity of MMP3 against collagen IV (Col IV) was measured using an EnzChek gelatinase/collagenase assay kit and fluorescently labeled DQ-type IV collagen. The x-axis represents the reaction time; the y-axis represents the relative fluorescence of degraded Col IV. The data represent the mean ± S.E.M. and were analyzed using two-way ANOVA (*P < 0.05). c A Transwell migration assay was conducted to confirm whether Col IV inhibited the migration of BMCs and d to confirm whether MMP3 accelerated the migration of BMCs in the presence of a Col IV barrier. Col IV was applied to the membrane inserts of a 24-well Transwell plate. BMCs were incubated with or without TMSC-CM for 4 h. The number of BMCs that migrated to the lower chamber was determined using 0.4% trypan blue staining. The data represent the mean ± S.E.M. and were analyzed using the Student’s t test (*P < 0.05)