Senescence Induction and PLLA Treatment in Fibroblasts

Human fibroblasts (CCD-986sk; American Type Culture Collection, Manassas, VA, USA) were maintained in Iscove’s Modified Dulbecco’s Medium (Welgene), 10% FBS (Gibco-Thermo Fisher Scientific), and 1% P/S (Welgene) in a 37 °C incubator with 5% CO₂. Senescence was induced by treating fibroblasts with 350 μM H₂O₂ for 1.5 h. Cells were washed with DPBS, and then incubated in fresh growth medium for 72 h to induce senescence [35 (link)]. Senescent and non-senescent fibroblasts were treated with PBS or 200 μg/mL PLLA for 48 h (Figure S2). After treatment, cell lysates were collected using a cell scraper for protein analysis. The fibroblasts used in this study were passaged 7–9 times, and the in vitro model was generated when the cells reached approximately 70% confluency.