β gal vector

Hec1a cells were cultured in 12-well-plates and transfected with 500 ng of pMir-3′UTRSMAD4 wt or mutated plasmid or pMir control vector together with 50 ng of β-GAL vector and 50 pmoles of pre-miR-205, pre-miR-146a, pre-miR-1260b, or pre-miR-negative control#1 (Thermo Fisher Scientific). Transfections were carried out using Lipofectamine 2000 and OPTI-MEM as recommended by the manufacturer (Thermo Fisher Scientific). At 48 h after transfection, luciferase activity was measured using the Luciferase Reporter Assay (Promega). Each transfection was repeated twice in triplicate. Transfection efficiency was corrected to β-GAL expression in all cases.

The 3′UTR of COL10A1 and MEF2C containing a seed region for miR-218 were amplified from genomic DNA by PCR. The 3′UTR fragment was cloned into SpeI and HindIII restriction sites of pMIR-REPORT Luciferase vector (ThermoFisher Scientific). The 3′UTR of RUNX2 containing a seed region for miR-218, as well as all mutated sequences, were chemically synthesized (GeneArt, Germany). Primers used for this assay are listed in Table S2.
For miR gain-of-function experiments, microRNA mimics and the miR-218 inhibitor (anti-miR-218) were purchased from Ambion (mirVana: MC10328, MH10328, 4464058).
HEK293T cells, seeded a day before transfection at a density 5 × 10⁴ cells/well, were cultured on 24 well plates. Co-transfection of 50 nM of a selected miR mimic combined with 250 ng of a corresponding tested reporter construct, together with 250 ng of a normalization control β-Gal vector (ThermoFisher Scientific), was carried out using Lipofectamine 2000 reagent (Invitrogen, Germany). Luciferase activity was measured 48 h after transfection with Victor3 Multilabel Counter 1420–042 using the Luciferase Reporter Assay Kit (Promega, USA). Luciferase intensity signals were normalized to the β-Galactosidase activity in the same sample. Three independent experiments with six biological replicates were performed for this assay.