Ahr sirna

siRNAs targeting AhR (AhR siRNA; Ambion; Thermo Fisher Scientific, Waltham, MA, United States) and a scrambled sequence control siRNA, which would not induce specific degradation of any cellular mRNA, were obtained from Ambion (Ambion; Thermo Fisher Scientific). HaCaT cells, cultured in 6-well plates, were incubated for 24 h in 0.5 mL of culture medium with a mixture containing 5 nM siRNA and 3 µL of Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific). Transfection was carried out after 24 h of culture using siRNA duplexes targeting the AhR mRNA sequence or control 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.