Figure 3. Gelatin zymography using conditioned media from primary vaginal stromal cells.

(A) Conditioned media were harvested from WT cells after 24 hours of stimulation. Fibronectin (FN) did not alter gelatinolytic activity in WT cells. TNF-α was used as positive control for MMP-9 induction. (B) Fbln5^–/– (KO) or Fbln5^RGE/RGE (R) cells were incubated with FN, fibulin-5 (F5), or both simultaneously (FN+F5). Note a marked upregulation of MMP-9 after fibronectin stimulation in mutant cells. Whereas fibulin-5 alone showed no effects, fibulin-5 inhibited fibronectin-induced upregulation of proMMP-9. (C and D) Incubation with β₁ integrin–blocking antibody or RGD peptides inhibited fibronectin-induced upregulation of MMP-9 in KO (C) and RGE (D) stromal cells. Experiments were performed 4 times using independent primary cell preparations. In B, the last 7 lanes were run on the same gel but were noncontiguous.