Fig. 4

ADAM8 promotes ERK activation, which is required for miR-720 expression. a Hs578T cells were cultured on plastic (two-dimensional (2D)) or in suspension (three-dimensional (3D)) for 48 h. WCEs and RNA were then isolated. WCEs were subjected to Western blotting for the ERK1/2 phosphorylated form (pERK1/2) and β-actin. The data from this and two other replicate experiments were quantified. The value of pERK1/2 in the 3D culture is given relative to the 2D culture, which was set to 1.0. b HEK-293 cells were transfected as above with empty vector (EV) or with vectors expressing ADAM8 (WT) or remnant form (Rem.) for 48 h. WCEs were analyzed for pERK1/2 and β-tubulin. The data from this and two independent replicate experiments were quantified. The value of pERK1/2 in the control EV DNA was set to 1.0. c, d MDA-MB-231 cells were cultured in serum-free medium for 24 h, and treated with 25 μM of ERK inhibitor FR180204 or control carrier DMSO for an additional 24 h. WCEs and RNA were collected. WCEs were subjected to Western blotting for pERK1/2 and β-tubulin (c). The data from this and two independent replicate experiments were quantified. The value of pERK1/2 in the DMSO sample was set to 1.0. RNA was subjected to RT-qPCR analysis and miR-720 levels were determined. The control condition (DMSO) was set to 1.0 (mean ± SD from three independent experiments) (d). e SUM-149 cells were cultured in serum-free medium for 24 h, treated with 25 μM of ERK inhibitor FR180204 or carrier DMSO as a control for an additional 24 h. RNA was subjected to RT-qPCR analysis for miR-720 levels. The DMSO control condition was set to 1.0 (mean ± SD from three independent experiments). **P < 0.01, ***P < 0.001, Student’s t test