Jetpei macrophage transfection reagent

After plating and differentiating THP-1 cells as described above, cells were transiently transfected with hERα-pSG5 (kindly provided by P. Chambon, Institut Clinique de la Souris, Illkirch Cedex, France), or with pcDNA flag ERα wt or pcDNA flag ERα C447A (kindly provided by F. Acconcia, University Roma Tre, Rome, Italy), and with pERE-TkGL3 (kindly provided by P.J. Kushner, Metabolic Research Unit and Diabetes Center, University of California, San Francisco, USA), and pRL-CMV (Promega), a renilla luciferase vector. Transfection was performed with jetPEI^®- Macrophage transfection reagent (Polyplus-transfection) according to the manufacturer’s instructions. Prior to stimulation, cells were starved for a maximum of 10 h. After harvesting, cell lysates were used to measure luciferase activity with the dual-luciferase reporter assay system (Promega).

Small interfering RNA (siRNA) for PHLDA1 and Tollip were designed and synthesized by GenePharma (Shanghai, China). The above siRNA fragments were transfected into RAW264.7, BMDM, or L-929 cells using jetPEI^®-Macrophage transfection reagent (Polyplus Transfection, Illkirch, France) or Lipofectamine RNAiMAX according to the manuals of the manufacturer. The above cells were further analyzed at 72 h after transfection. PHLDA1 and Tollip siRNA sequences are listed in Supplementary Table 2. Plasmids were transfected into RAW264.7, BMDM, L-929, or 293T cells using Lipofectamine 2000, jetPEI^®-Macrophage transfection reagent, or EZ Cell Transfection Reagent II according to the manuals of the manufacturer. The above cells were further analyzed at 48 h after transfection.