Fig. 2

miR-720 is secreted in media and its expression is positively regulated by ADAM8 in TNBC and HEK-293 cells. a Cultures of MDA-MB-231 and SUM-149 cells at 50–70 % confluency were grown either in serum-free or exosome-depleted FBS media, respectively. After 72 h, supernatants were collected, exosomes purified, and RNA isolated and subjected to RT-qPCR analysis for miR-720. miRNA expression levels are given as mean ± SEM from two independent experiments. b Exosomes were purified from control shRNA (shCtrl-3) and a stable clone of MDA-MB-231 cells expressing ADAM8 shRNA (shA8-20) and relative miR-720 expression levels determined, as described for MDA-MB-231 cells in part (a). miR-720 levels in exosomes released by the two lines are given as the mean ± SEM from two independent experiments. c–f MDA-MB-231 shA8-20 and HEK-293 cells were transfected in six-well plates with 4 or 2 μg, respectively, of empty vector (EV) or an ADAM8 expression vector (A8) for 48 h. WCEs from shA8-20 (c) and HEK-293 (d) cells were subjected to Western blotting for ADAM8 and β-actin, as above. Representative blots are shown (n = 3). RNAs from shA8-20 (e) and HEK-293 (f) cells were subjected to RT-qPCR for miR-720 levels, as in Fig. 1 f. Control condition (EV) is set to 1 (mean ± SD from three independent experiments). g, h Hs578T cells were cultured on plastic (two-dimensional (2D)) or in suspension (three-dimensional (3D)) for 48 h, and WCEs and RNA isolated. WCEs were analyzed by Western blotting for ADAM8 and β-actin (g), and RNA subjected to RT-qPCR for miR-720 levels as above (h). EV is set to 1 (mean ± SD from three independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test