Mission mirna

Smooth muscle cells were isolated from cystectomized human bladders as described [4 (link)],[24 (link)]. Cells in passages 2–6 were cultured in DMEM/Ham’s F12 supplemented with antibiotics (penicillin and streptomycin) and 10% fetal calf serum (FCS). The cells were treated for 24h with the following agents: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 0.16 μM 48599, Sigma Aldrich), forskolin (10 μM, F6886, Sigma Aldrich), brefeldin A (5 μg/ml, B7651 Sigma Aldrich), tunicamycin (5 μg/ml, T7765, Sigma Aldrich), Phorbol 12-myristate 13-acetate (PMA, 1μM, P8139, Sigma Aldrich) or fetal calf serum (FCS). Cells were harvested and RNA isolated as described above. MiR-132 and miR-212 inhibitors (AM10166, AM10340, Ambion, Thermo Scientific, Pittsburgh, PA, USA; 10 nM and 100 nM), mimics (Mission miRNA: Sigma-Aldrich, St. Louis, MO, USA; 10 and 100 nM) and negative control (HMC0002, Mission miRNA: Sigma-Aldrich, St. Louis, MO, USA; 10 and 100 nM) were transfected using Oligofectamine reagent (Life Technologies). Successful transfection of miR-132/212 mimics and inhibitors was confirmed using real-time quantitative PCR. Experiments with human cells are from 3–7 independent replicates using cells from both males and females.

Cells were cultured on collagen-coated plates in growth media, consisting of 40% (v/v) low-glucose DMEM, 40% (v/v) HAM's F-10 medium, 20% fetal bovine serum, 5 ng/ml basic fibroblast growth factor, and 100 U/ml Penicillin/Streptomycin (all from Life Technologies). To induce differentiation, myoblasts were transferred to differentiation media (low-glucose DMEM supplemented with 2% horse serum and 100 U/ml Penicillin/Streptomycin) after reaching 80% confluency. Lipofectamine RNAiMax was used for transfecting miRNA mimics (MISSION miRNA, Sigma-Aldrich) or antagomirs to the cells. Final concentration was 38 nM for mimics and 12 nM for antagomirs, respectively. Lipofectamine LTX was used for DNA transfection, and co-transfection of DNA with miRNA mimics or antagomirs was performed using Lipofectamine 2000. All reagents were purchased from Invitrogen, and transfection was performed according to the manufacturer's instructions. MG-132 (474790) was obtained from Merck Millipore. HEK 293 cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum and 100 U/ml Penicillin/Streptomycin (all from Life Technologies). Lipofectamine 2000 (Invitrogen) was used for co-transfection of DNA with miRNA mimics or antagomirs, according to the manufacturer's instructions.

miRNA mimics (hsa-miR-34b^*,−222,−320d,−484, and scrambled control; MISSION miRNA, Sigma-Aldrich, #HMI0510, #HMI0400, #HMI0475, #HMI0593, #HMC0002) were reverse transfected into HEK293T cells (2.5 × 10⁵ cells in a 6-well plate) using the Lipofectamine RNAiMAX transfection reagent (ThermoFisher Scientific, #13778075) as recommended by the supplier. Mimics were used at a final concentration of 50 nM. To examine potential synergistic effects, a combination of all four miRNA mimics (12.5 nM each) was compared with scrambled controls (50 nM) or miR-34b^* mimic (12.5 nM) only. The latter was co-transfected with scrambled control mimics (37.5 nM) to ensure comparable transfection conditions.