Siarnt

HaCaT keratinocytes and normal human dermal fibroblasts (NHDF) were a gift from Luis Garza (Johns Hopkins University). Anti–β-actin (ab8227) and anti-MMP2 (WB: ab97779; IHC: ab86607) antibodies were purchased from Abcam. Anti-MMP11 antibody (NBP2-67670) was purchased from Novus. Anti–phospho-SP1 (Thr-278: PA5-106039; Thr-739: PA5-104771), HRP-conjugated anti–rabbit IgG (A27036), HRP-conjugated anti–mouse IgG (A28177), and A488-conjugated anti-mouse (A11029) antibodies were purchased from Invitrogen. Anti-AhR (83200S), anti-ARNT (5537S), anti-SP1 (9389S), anti-ATM (2873T), anti-γH2AX (2577S), and normal rabbit IgG (2729S) antibodies were purchased from Cell Signaling Technologies. CH223191 (C8124), vitamin B₁₂ (V2876), and FA (F7876) were purchased from Sigma-Aldrich. PD98059 (9900S), SB203580 (5633S), and SP600125 (8177S) were purchased from Cell Signaling Technologies. The following siRNAs were purchased from Thermo Fisher: siNC (catalog 4390843), siAHR (catalog 4390824, s1199), siARNT (catalog 4392420, s1615), siSP1 (catalog 4392420, s13319), and siATM (catalog 4392420, s57221).

For transfection, isolated BMDMs were differentiated with 20 ng/ml M-CSF for 1 week and transfected afterwards with 150 nM SMARTpool ON-TARGET plus siArnt or ON-TARGET plus NON-TARGETing pool siCtrl (both Thermo Scientific, Karlsruhe, Germany) using Hiperfect transfection reagent (Quiagen, Hilden, Germany). Therefore, BMDMs were incubated in medium without FCS containing siRNA and Hiperfect. After 6 h, medium containing M-CSF (final concentration of 20 ng/ml) was added and after 24 h cells were stimulated with MCA (5 μg/ml) or dimethylsulfoxide (DMSO) as a control for 8 h.