Viromer blue transfection reagent

To confirm the effect of blocking AhR signaling on regulating c-fos and NFATc1 expression, genetic knock down of AhR was performed. In Brief, Raw 264.7 cells at density of 4 × 10⁴ cells/well were seeded in 12 well plate and allowed to attach overnight. Next day, cells were transfected using Viromer Blue transfection reagent (#TT100300, OriGene Technologies, Rockville, MD, USA) and Ahr Mouse Small interfering RNA (siRNA) Oligo Duplex (AhR si, # SR420301C, OriGene Technologies, Rockville, MD, USA) at a final concentration of 10 nM. Trilencer-27 Universal scrambled negative control siRNA duplex was used to transfect control groups at a final concentration of 10 nM. One or two days after transfection, cells were treated with KYN (10 or 25 μM) with or without RANKL (20 ng/mL) for 24 h before collecting the cells for further Western blotting analyses.

Human fibroblasts were cultured in 96-well plates. After 24 h, we differentiated fibroblasts into myofibroblasts by incubation with TGFβ for three days, with re-stimulation with TGFβ every twenty-four hours. We then treated the cells with CQ (10 µM, Biotrend) or 3-MA (5 mM, Sigma-Aldrich). Alternatively, we knocked down ATG7 and BECLIN1 (50 nM) (pre-designed, Invitrogen, CA, USA), via siRNA transfection using Viromer Blue transfection reagent (OriGene Technologies, Rockville, MD, USA), and continued the stimulation with recombinant TGFβ for two additional days. The control group (Fib) was pre-treated with vehicle for three days, then transfected with non-targeting siRNA and treated with vehicle for additional two days. The deactivated myofibroblast group (deact MyoFib) was pre-treated with TGFβ for three days, then transfected with non-targeting siRNA and treated with vehicle for two days. Afterwards, cells were fixed with 4% PFA and immunostained for stress fibers and αSMA as described above (immunohistochemistry and immunofluorescence staining section). Images were captured using CellInsight CX5 High-Content Screening (HCS) Platform (Thermo Fisher Scientific; software HCS Navigator Version 6.6.1). Data were analyzed using HCS Studio Cell Analysis Software 6.6.1 (Thermo Fisher Scientific).