Mir 3085 3p mimic

SW1353 cells were plated in 60-mm culture dishes at 3.5 × 10⁵ cells/dish overnight. The cells were then transiently transfected with either miR-3085-3p mimic (Qiagen) or non-targeting control (Qiagen), serum starved, and treated with IL-1β or TGFβ1 as described above. Nuclear and cytosolic fractions were purified using the Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific) according to manufacturer’s instructions, using 1 × protease inhibitor cocktail III (Thermo Fisher Scientific), 1 × phosphatase inhibitor cocktail 2 (Sigma Aldrich), and 1 × phosphatase inhibitor cocktail 3 (Sigma Aldrich).

SW1353 cells were plated in 6-well plate wells at 1.5 × 10⁵ cells/well and left to adhere overnight. Cells were transiently transfected with 50 nM miR-3085-3p mimic (Qiagen), siRNA (Qiagen) or non-targeting controls (Qiagen) for 48 h. After serum starvation for another 24 h, cells were stimulated with IL-1β (5 ng/ml) (First Link (UK) Ltd) for 30 min or TGFβ1 (4 ng/ml) (R&D Systems) for 2 h and washed twice in ice-cold phosphate buffered saline (PBS). Whole cell lysates were harvested into ice cold RIPA buffer (50 mM Tris–HCL pH7.6, 150 mM NaCl, 1% (v/v) Triton x-100, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 10 mM NaF, 2 mM Na₃VO₄, 1 × protease inhibitor cocktail III (Thermo Fisher Scientific)). Samples were separated on reducing SDS-PAGE, transferred to PVDF membrane and probed overnight at 4 °C. The p65 (#8242), phospho-p65 (#3033), IκBα (#4814), phosphor-IκBα (#2859),SMADs (#3103, #9513, #38,454), phospho-SMADs (#9523), MyD88 (#4283), β-catenin (#9582), phospho-β-catenin (#9561) and GAPDH (#2118) (all from Cell Signaling Technology, used at recommended concentrations) were detected using HRP-conjugated secondary antibodies (DAKO), visualised using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific), and imaged by ChemiDoc MP Imaging System (Biorad)^{13 (link)}.