Sp1 sirna

Luciferase assays were performed according to the manufacturer's instructions (Promega CAT.# E2510). The human MMP9 promoter Luc reporter which construced by human MMP9 promoter (−700,−100) and Luc reporter construct (PGL 4.20) (Addgene, Cambridge, MA) was developed by Dr Zhuang’ lab (SNB, NINDS, NIH). The Sp1 siRNA (Cat. # sc-90024, Santa Cruz) and PC4 siRNA (Cat.# sc-38583, Santa Cruz). Transfections were performed using Lipofectamine 2000 (Cat. #12566014, Invitrogen). Normalizing the DNA amount in each well with empty vector DNA, and the luciferase activity was normalized to light units per microgram protein. Experiments were performed in triplicate.

Human primary dermal fibroblasts (HDFs) (American Type Culture Collection (ATCC); Manassas, VA, USA) were maintained in fibroblast basal medium (ATCC) supplemented with fibroblast growth kit low serum (ATCC)^{39 (link)}. Cells were incubated with recombinant human TGF-β1 (10 ng/mL; R&D Systems, Minneapolis, MN, USA) and a combination of the following: TLY012^{13 (link)}, anti-DR4 Ab (Mapatumumab; Creative Biolabs, Shirley, NY, USA), anti-DR5 Ab (Conatumumab; Creative Biolabs), Smad 2 inhibitor SB203580 (Selleckchem, Houston, TX, USA), Smad3 inhibitor SIS3 (Selleckchem), or MAPK inhibitor PD98059 (Selleckchem). For gene silencing studies, cells were transfected with DR4 siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), DR5 siRNA (Santa Cruz Biotechnology, Inc), Smad2/3 siRNA (Santa Cruz Biotechnology, Inc.), SP1 siRNA (Santa Cruz Biotechnology, Inc.) or Smad2/3 shRNA (Santa Cruz Biotechnology, Inc.) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Human dermal fibroblasts from patients were generated as previously described^{20 (link)}. Skin biopsies from three patients were digested using dispase II (Sigma-Aldrich, St. Louis, MO, USA) with 10% heat-inactivated FCS and cells were maintained in DMEM/F-12 medium. All cell lines were negative for mycoplasma.