Figure 3

Immunodepletion of SPARC abrogates the M pro-invasive activity of S-conditioned medium. (a) Immunodepletion of SPARC abrogates the ability of S conditioned medium to enhance the invasiveness of M cells. Top panel: Western blots show the amount of SPARC protein present in immunoprecipitates (IP) and supernatants (Sn) of S conditioned medium subjected to immunoprecipitation of SPARC using 2 μg/mL of anti-SPARC plus protein G (2 h incubation at 4°C). IgG plus protein G was used as a specificity control. C corresponds to control medium and (-) corresponds to S-CM treated only with protein G beads but no specific antibodies. Bottom panel: Relative invasiveness in Transwell-Matrigel assays of M cells treated with control medium, S-conditioned medium, and medium immunodepleted for SPARC or its IgG control. (b) Same as in a, substituting a specific anti-SPARC antibody for anti-PAI-1. (c) Exogenous SPARC stimulates the invasiveness of M cells. Transwell-Matrigel assay comparing the relative invasiveness of M cells under standard conditions or treated with 20 μg/mL of recombinant human SPARC protein. All experiments were performed in quadruplicates or triplicates and the data shown are percentages of invasiveness relative to control ± SD. Asterisks denote statistically significant differences (two-tailed Student’s t-test).