Pre mir125b 5p

To generate MMP11 luciferase reporter constructs, fragments of putative miR125b-5p binding sites were cloned into the 3′UTR region using HindIII-SpeI restriction sites of the pMIR-report plasmid (Ambion, Carlsbad, USA) (Fig. 2D). Correct insertion was confirmed by sequencing. For dual luciferase assays, 3×10⁵ SGBS cells/ml were co-transfected with 0.2 µg of firefly luciferase reporter plasmid, 0.1 µg of a control vector containing Renilla luciferase (Promega, Fitchburg, Madison, USA), and 500nmol of pre-miR125b-5p (Ambion) or scrambled negative control RNA (pre-ntRNA, Ambion) 24h post-transfection. Luciferase activity was quantified using Dual Luciferase Assay System (Promega) according to the manufacturer's instructions. Putative miR125b-5p binding sites were mutated using site-directed mutagenesis primers.
MMP11 mutagenesis Primer (1): F5′-TGGCTTGGATGCCCTAATGAGTGCTGACCCCTGCC-3′, R5′-GGCAGGGGTCAGCACTCATTAGGGCATCCAAGCCA-3′; MMP11 Mutagenesis Primer (2): F5′-CTGAGCCCATGTCTCCTAATGAGGATGGGGTGGGGT-3′, R5′-ACCCCACCCCATCCTCATTAGGAGACATGGGCTCAG-3′; MMP11 Mutagenesis Primer (3): F5′-TCTCCATCCCTGTCCCTAATGATAGCACCATGGCAGGAC-3′, R5′-GTCCTGCCATGGTGCTATCATTAGGGACAGGG ATGGAGA-3′.

Preadipocytes were transfected using the Neon Transfection System 100 μl Kit (Invitrogen) according to Bernhard et al.⁴³ (link) For MMP11 overexpression, 1.25 µg of expression plasmid (Ambion, Abingdon, UK) were transfected and empty pCMV6-XL5 vector was used as a control. For miR125b-5p overexpression, 500 nmol of pre-miR125b-5p (Ambion) or a scrambled negative control (pre-ntRNA, Ambion) were transfected. For MMP11 and PPARg knockdown experiments, gene-specific ON-TARGETplus SMARTpool small interfering (si)RNAs and ON-TARGETplus control reagents (Dharmacon, Lafayette, LA, USA) were used at a final concentration of 500 nmol/l. The downregulation of miR125b-5p was carried out by transfection with antagonizing miR125b-5p (anti-miR125-5p, Ambion) or scrambled negative control RNA (anti-ntRNA, Ambion). After electroporation 350,000 cells were seeded in 6-well format and differentiated. Efficient knockdown was confirmed on day 0 and 12 post-induction by qRT-PCR.