A Cell-penetrating Peptide Suppresses Inflammation by Inhibiting NF-κB Signaling

Yu Fu Wang1,2§, Xiang Xu1§, Xia Fan1, Chun Zhang1, Qiang Wei1, Xi Wang1, Wei Guo1, Wei Xing1, Jian Yu3, Jing Long Yan2, Hua Ping Liang1

1State Key Laboratory of Trauma, Burn and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, PR China. 2Department of Orthopedics, First Affiliated Hospital of HarbinMedicalUniversity, Harbin, PR China

3Department of Pathology, University of Pittsburgh, PA15213, USA

§These authors contributed equally to this work.

Correspondence should be addressed to: Hua-Ping Liang, The State Key Laboratory of Trauma, Burn and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, 10 Branch Road, Changjiang Street, Chongqing 400042, PR China Phone: 023-68757411 Fax: 023-68757404 E-mail: or Jing-Long Yan, Department of Orthopedics, First Affiliated Hospital of Harbin Medical University, 23 Youzheng Street, Nangang District, Harbin, 150001, PR China Phone: 0451-85555155 Fax: 0451-85555155 E-mail:

Supplemental Materials and Methods

Chemicals and MaterialsFetal bovine serum (FBS) and other culture materials were purchased from Invitrogen (Carlsbad, CA, USA). Transcription Factor Assay Kits, Jurkat nuclear extracts, the recombinant NF-κB p65 protein and recombinant NF-κB p50 protein were purchased from the Active Motif Company (Carlsbad, CA, USA). Jurkat nuclear extracts were collected from Human T lymphoblast which were cultured in medium supplemented with 50 ng/ml TPA (phorbol, 12-myristate, 13 acetate) and 0.5 µM calcium ionophore A23187 (CI) for two hours at 37°C immediately prior to harvesting. Rabbit anti-IB, Rabbit anti-IBβ, Rabbit anti-IBε, Rabbit anti-p-IB, Rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Rabbit anti-NF-κB p65 antibody and FITC-labeled secondary antibody were all bought from Santa Cruz Biotechnology, CA, USA. Anti-p50 antibody and anti-p65 antibody were also from Santa Cruz Biotechnology. phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA) and cytokine IFN- were from Sigma-Ardrich,St. Louis, USA. TNF-, IL-1, and IL-6 ELISA kits were purchased from Wuhan Boster, Wu Han, PR China. PGE2 and NO ELISA kits were purchased from R&D Systems, MN, USA. WST-1 was obtained from Beyotime institute of Biotechnology, Jiang Su, PR China. Zymosan (S. cerevisiae) was purchased from Sigma-Aldrich, St. Louis, USA.

Luciferase reporter assays RAW 264.7 cells were plated at a density of 0.5×105 per well in 24-well plates and allowed to grow up to 70-80% confluence. RAW 264.7 cells were transfected with pAP-1,pSTAT1/3 and p4-Bluciferase reporter vectorusing Lipofectamine 2000 (Invitrogen) for 6 h,respectively.After washing with fresh complete medium, these cells weretreated with AIP6 or NCP (150 M) for 24 h.Then the cells harboring pAP-1 or p4-Bluciferase reporter vector werestimulated withphorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA) (50 ng/ml) for 16h, and the cells harboring pSTAT1/3luciferase reporter vectorwere stimulated with IFN-(20 U/ml) for 16 h. These cells were harvested for detection of luciferase activity and transcriptional activity is represented as relative luminescence units (RLU).