Arnt sirna

AHR siRNA (siRNA ID number: s1199), ARNT siRNA (siRNA ID number: s1613) and MMP1 siRNA (siRNA ID number: s8847) were purchased from Ambion’s Silencer Select pre-designed siRNA library (Grand Island, NY). A non-specific, negative control siRNA (negative control #1, Ambion) was used as the control. Cells were grown to 70–80% confluence in 6-well plates and treated with the siRNAs mixed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in OptiMEM I (Invitrogen, Carlsbad, CA) at a final concentration of 50 nM for 24 hours, according to the manufacturers’ instructions. Cells were then treated with or without TGFβ and/or FICZ as described.

Gene silencing was achieved by a reverse transfection procedure using Lipofectamine® RNAiMAX (Life technologies) in accordance with the manufacturer’s guidelines. Therefore, cells were seeded on 24-well plates and mixed with the transfection mixture containing either ARNT siRNA (#s1613, Ambion) or BLOCK-iT™ Fluorescent Control siRNA (#442926, Life technologies). Subsequently cells were incubated overnight and subjected to irradiation. To gain protein lysates for Western blot analysis, the transfection procedure was performed in 6-well plates.