Figure 4. Lumican inhibits MSC migration and invasion.

MSC (A) or EPC (B) were plated on 12-well plates at 15 000 cells per chamber of culture-insert. Cells were incubated at 37°C and 5% CO₂ for 24 h. After withdrawal of the culture-insert, lumican (100 nM) was added to the serum-free cell culture medium. The migration speed of MSC and EPC was determined by means of computer-assisted phase contrast videomicroscopy during 48 hours as described in Materials and Methods. Representative images of cell positions after 48 h of migration are displayed on the right panels. The data are representative of three independent analyses. *p<0.05. (C) MSC and EPC (D) invasion in presence of lumican. Cells were seeded on inserts as described in the Materials and Methods section. Medium with 10% or 2% FBS was used as a chemotactic agent for MSC or EPC, respectively, and the cells were cultured for a further 48 h. Negative control medium contained 2% BSA. Medium containing 0.5% BSA and 100 nM lumican was added to the upper chamber at the time of seeding. Invading cell nuclei were stained with Hoechst 33342 (5 µg/mL) and counted using fluorescence microscopy. Representative fluorescence images of invading cells are displayed on the upper panel. Results are expressed as the percentage of control (cells invading toward chemotactic medium) (mean ± S.D.) from at least three independent experiments with triplicate inserts. Scale bar: 100 µm. **p<0.01; (E, upper panel): Lysates from transiently transfected MSC with GFP (mock) or MMP-14-GFP constructs were analyzed for MMP-14 overexpression by Western blotting with an antibody raised against MMP-14 after 48 h of transfection. Non-transfected MSC lysate is also included as control. (E, bottom panel): Overexpression of MMP-14 in MSC induces MMP-14 activity as shown by the enhancement of active MMP-2 band by zymography. (F): Migration of GFP- (mock) or MMP-14-GFP-transfected MSC was determined 48 h post-transfection using cell culture-insert assay. The migration speed of GFP-positive MSC, was determined by means of computer-assisted phase contrast and fluorescence videomicroscopy during 24 hours as described in supplemental methods section. The data are representative of two independent analyses. **p<0.01. (G): GFP- (mock) or MMP-14-GFP-transfected MSC invasion in presence of lumican. Cells were cultured and inserts seeded as described in the supplemental methods section. Medium with 10% FBS was used as a chemotactic agent for MSC, and the cells were cultured for a further 48 h. Negative control medium contained 2% BSA. Medium containing 0.5% BSA and 100 nM lumican was added to the upper chamber at the time of seeding. Invading cell nuclei were stained with Hoechst 33342 (5 µg/mL) and counted using fluorescence microscopy. Representative fluorescence images of invading cells are displayed on the upper panel. Results are expressed as the percentage of control (mock-transfected cells invading toward chemotactic medium) (mean ± S.D.) from two independent experiments with triplicate inserts. Scale bar: 100 µm. *p<0.05. ***p<0.001.