Supplementary data for Fig 3.

<div><p>Gingival overgrowth caused by cyclosporine A is due to increased fibroblast proliferation in gingival tissues. Cell cycle system balances proliferation and anti-proliferation of gingival fibroblasts and plays a role in the maintenance of its population in gingival tissues. When cells detect and respond to abnormalities (e.g. DNA damage), cell cycle progression is arrested in the G<sub>1</sub> phase until the completion of damage restoration. In this study, we investigated the effects of cyclosporine A on G<sub>1</sub> cell cycle arrest and on its regulators in gingival fibroblasts to clarify the mechanism of cyclosporine A-induced gingival overgrowth. Human gingival fibroblasts from healthy donors were cultured to semi-confluence and were then treated with or without 200 ng/mL (166 nM) cyclosporine A in D-MEM with 2% fetal bovine serum. Cell proliferation was assessed by counting total cell numbers. The distribution of cell cycle phases was assessed using flow cytometric analysis. The levels of mRNA and protein expression for cell cycle regulators were quantified using reverse transcription-quantitative PCR and western blot analysis, respectively. Treatment with cyclosporine A markedly increased cell proliferation, inhibited G<sub>1</sub> cell cycle arrest, significantly increased <i>CDC25A</i> and <i>CYCLIN E1</i> mRNA expression levels, significantly decreased <i>P21</i>, <i>SMAD3</i> and <i>SMAD4</i> mRNA expression levels, significantly upregulated the protein expression levels of CDC25A, CYCLIN E1, pCDK2 and pRB1 and significantly downregulated the protein expression levels of P21, SMAD3 and SMAD4. Treatment with cyclosporine A also increased <i>MYC</i> and <i>ATM</i> mRNA expression levels and decreased <i>CDK2</i>, <i>ATR</i>, <i>P27</i>, <i>P53</i> and <i>RB1</i> mRNA expression levels but not significantly. These results demonstrate that cyclosporine A causes gingival overgrowth due to the following mechanism in gingival fibroblasts: cyclosporine A increases levels of phospho-CDK2 and CYCLIN E1 by upregulating CDC25A and downregulating P21 with the downregulation of SMAD3 and SMAD4, which results in the inhibition of G<sub>1</sub> cell cycle arrest.</p></div>