Silencer select pre designed sirna library

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.

The validated mouse Pref-1 siRNA#1 (s64996, antisense region located at exon 6 of Pref-1), Pref-1 siRNA#2 (s64998, antisense region located at exon 3 of Pref-1) and a non-targeting siRNA as the negative control were obtained from Ambion Silencer Select Pre-designed siRNA library (Ambion). Two distinct exon targets of siPref-1 were applied for verifying that the effect was not due to the siRNA sequence specificity. Following the manufacturer's instructions, on the previous day, 3T3-L1 pre-adipocytes were seeded in six-well plates at approximately 60% confluence (6 × 10⁵ cells per well). The siRNAs at f.c. of 100 nM (in Opti-MEM) were pre-mixed with 5 μl lipofectamine 2000 (Invitrogen) in 500 μl Opti-MEM medium (Invitrogen,). Then the mixture was added into each well. Six hours post transfection, the medium containing siRNA was replaced with fresh medium. Two days post confluence, the transfected cells were induced to differentiate as described above.