Allstars negative sirna control

The day before transfection, SW1736 cells were plated in a 6-well culture plate at density of 2 × 10⁵ cells per well. Cells were transiently transfecting with Roche’s XtremeGene siRNA transfection reagent (Roche, Basel, Switzerland); with 160 pmoles of FOXE1/ELK1 specific siRNA or AllStars negative siRNA control (Qiagen). Forty-eight hours after transfection, total RNA was extracted using an RNeasy Plus mini kit (Qiagen).

Knee SFs were transfected with 25 nM siRNA targeting HOTAIR (Hs HOTAIR 3 siRNA, Cat. No. SI04446036, Qiagen, Sequence: 5′-CACGGAACCCATGGACTCATA-3′) or 50 nM antisense LNA HOTAIR GapmeR (Cat. No. 300600, Design ID 542251-1, Exiqon, Sequence: 5′-AGGCTTCTAAATCCGT-3′) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. AllStars Negative siRNA control (25 nM, Qiagen) or Antisense LNA GapmeR Negative Control A (Cat No 300610) were used as transfection controls. At 24 h after transfection the medium was replaced and cells were lysed 48 and 72 h after GapmeR and siRNA transfection, respectively. In TNF stimulation experiments, 48 h after transfection with siRNA, SFs were stimulated with TNF (10 ng ml⁻¹, R&D Systems, 210-TA-010) for additional 24 h, before supernatants were collected and RNA isolated for enzyme-linked immunosorbent assay (ELISA) and real-time PCR analysis. Transfection with siHOTAIR and HOTAIR GapmeR reduced the expression of HOTAIR in SFs for 48±21% (mean±s.d.) and 92% (median; min 62%—max 94%), respectively (Supplementary Fig. 6).