Fig. 3

ADAM8 DI domain induces expression of miR-720 via β1-integrin signaling. a, b HEK-293 cells were transfected in six-well plates with 2 μg of either pcDNA3.1 myc-his vector (EV-3.1), or vectors expressing ADAM8 (WT-3.1 or EQ-3.1) for 48 h. WCEs and RNA were then collected. Samples of WCEs were subjected to Western blotting for ADAM8 and β-actin, as in Fig. 1. A representative blot is shown (n = 3) (a). RNAs were subjected to RT-qPCR for measurement of miR-720 levels and values presented relative to the control condition (EV-3.1), which is set to 1 (mean ± SD from three independent experiments), as above (b). c, d HEK-293 cells were transfected as above with EV DNA or with vectors expressing ADAM8 WT or remnant form (Rem.) for 24 h and 48 h. WCEs harvested 48 h after transfection were analyzed for ADAM8 and β-tubulin. A representative blot is shown (n = 3) (c). RNA was subjected to RT-qPCR for measurement of miR-720 levels. EV control condition is set to 1 (mean ± SD from three independent experiments) (d). e HEK-293 cells were transfected in 12-well plates with a vector expressing the ADAM8 remnant form (Rem.). After 24 h, the transfected cells were treated with 20 μg/ml ADAM8 antibody MAB10311, which targets the CRD/ELD domains and inhibits DI activity [24], or isotype-matched control IgG1 for another 24 h. RNA was extracted and subjected to RT-qPCR analysis for miR-720 levels. Control condition (EV + IgG1) is set to 1 (mean ± SD from three independent experiments). f MDA-MB-231 cells were treated with 10 or 20 μg/ml β1-integrin (Anti-β1-integ.) antagonist antibody or isotype-matched control IgG2A for 24 h, and miR-720 levels determined by RT-qPCR analysis. Control condition (IgG2A) is set to 1 (mean ± SD from three independent experiments). **P < 0.01, ***P < 0.001, Student’s t test. Not sig. not significant, Rel. relative