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High‐throughput assays for gingival fibroblast in vitro wounds treated with nicotine, and/or TGFβ1
Author(s) -
Svoboda Kathy K H,
Cline Rob,
Cowan Cameron
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.582.4
Subject(s) - fibroblast , nicotine , phosphorylation , wound healing , p38 mitogen activated protein kinases , in vitro , transforming growth factor , chemistry , kinase , alkaline phosphatase , fibroblast growth factor , microbiology and biotechnology , andrology , protein kinase a , medicine , immunology , biochemistry , biology , receptor , enzyme
Nicotine treated gingival fibroblast cells have inhibited wound and TGFβ1 responses leading to altered phosphorylation of signaling proteins. Over 600 signaling proteins were screened for changes in phosphorylation when treated with nicotine and/or TGFβ1 and wounded in vitro and a high‐throughput cell culture assay was developed to screen effects on wound closure. Primary human fibroblasts were grown to confluence in 30mm dishes and 96 well plates, starved for 24 hours and treated with nicotine or TGFβ1 2 hours before wounding. Four treatment groups: controls (C), nicotine (N), TGFβ1 (TGF), and N+TGF were divided into wounded and unwounded groups. Cells were lysed in ice cold buffer with phosphatase inhibitors, frozen and transported to Kinexus for phosphorylated protein screening. A 96 well plate pin array was used to uniformly wound each well. Plates were fixed and stained at 0, 4, 7, 14, and 24 hours. Plates were imaged with a light scanner at 1200 dpi. Wound areas were measured with Metamorph. Proteins involved in cell migration including p38 MAP kinase, MEK1, and PCTK1 were increased in C and TGF, but decreased in N and TGF treated groups. N treatment increased phosphorylation of CDK1/2 and PKCd. The pin array wounded the cells in a uniform and predictable manner. Wounds had closed 50–56% by 4hr. Nicotine treated wounds were larger at 7 and 14 hr and closed at 24 hr.