Murine il 17a

After 24 h of starvation with HITES medium containing 0.1% FBS, MLE-12 cells were cultured with different concentrations (0, 1, 10 and 100ng/ml) of mouse recombinant murine IL-17A (R&D Systems, Minneapolis, USA) for 24h, and the expression of miR-365-3p was examined by qRT-PCR. Alternatively, the mimic (100nM) of miR-365-3p was transfected into the MLE-12 cells using X-tremeGENE siRNA Transfection Regent (Roche, Penzberg, Upper Bavaria, Germany) in Opti-MEM (Gibco) for 24h. Expression of ARRB2 was examined by qRT-PCR and Western blot.
We cultured 4 kinds of cells for MiR-365-3p transfection, including MLE-12, J774a.1, U937 and A549. the cells were transfected with the mimic (100nM) of miR-365-3p for 6h, and 24h later, the transfected cells were incubated with 10ng/ml murine IL-17A or 100 ng/ml human IL-17A (R&D Systems). RNA samples were collected 3h after the IL-17A stimulation. qRT-PCR were performed to evaluate the level of cytokines in IL-17A treated cells.

Hind paws were dissected under sterile conditions and the skin, tendons, and toes were removed. Primary FLS were isolated from hind paws by enzymatic digestion with 1 mg/ml Collagenase IV (Worthington, Lakewood, USA). Isolated FLS were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (Sigma-Aldrich, Darmstadt, Germany) supplemented with 10% heat-inactivated FCS (Biochrom GmbH, Berlin, Germany). FLS were used in passages 3–5. FLS (5 × 10⁴ cells/ 24-well) were stimulated with murine myostatin (100 ng/ml), murine IL-17A (20 ng/ml) or with a combination of both (R&D Systems, Nordenstadt, Germany) for 48 h. Supernatants were collected and either used for assessment of CCL20 secretion or as conditioned medium (CM) in T-cell migration assays.