MMP14 knockdown partially inhibits the shedding of CD13 from FLS.

<p><b>(A)</b> MMP14 mRNA was almost completely removed by transfection with MMP14 siRNA. ADAM10, ADAM15, and ADAM17 knockdown constructs each also almost completely removed their respective mRNAs with appropriate siRNA transfection. None of the siRNAs (MMP14, ADAM10, ADAM15, or ADAM17) had an off target effect on MMP1. n = 3 (<b>B)</b> Shedding of CD13 into FLS culture supernatant was inhibited by knockdown of MMP14. Further inhibition was seen with GM6001. CD13 in FLS lysate was not changed by knockdown of MMP14, and significantly increased in GM6001 treated FLS. FLS were grown to confluence then transfected with siRNA using an Amaxa nucleofector kit. Cells were grown to 75% confluence then switched to serum free growth media (Peprogrow) supplemented with 1ng/ml IL-1 and 10ng/ml TNFα for 48 hours with or without 25μM GM6001. <b>(C)</b> Cells were incubated for 1hour at room temperature with anti-CD13-FITC (1D7) 1μg/100μl and anti-MMP14-PE (128527) at 1.67μg/100μl. Figures shown were taken at 1000x. The four panels show: top left, MMP14 alone (red); top right, CD13 alone (green); bottom left, co-localization (co-localization in yellow); and bottom right, co-localization analysis by ImageJ (white = overlapping red and green pixels). All images were background corrected using DAPI alone and MsIg-FITC and MsIg-PE for threshold limits. Representative of n = 6 *p≤0.05 **p≤0.005 ***p≤0.0001 mean±SEM (n≥2)