Characterization of TIMP-1 Knockdown in NSCLC

Human NSCLC cell lines (A549 and H460) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). A549- and H460-derived cell clones, encoding non-target scrambled shRNA (-NT) or TIMP-1-specific knockdown shRNA (-KD) sequences, characterized in our previous studies [13 (link)] were used. A549 and its clones were cultured in an F-12K medium, and H460 and its clones were grown in RPMI Medium 1640 (Sigma-Aldrich, St. Louis, MO, USA) as per ATCC recommendations. Cells were cultured under normoxic (20% O₂ with 5% CO₂) or hypoxic conditions as indicated (1% O₂ with 5% CO₂). To generate knockdown clones of TIMP-1, we constructed TIMP-1 KD and NT clones with shRNA Lentiviral Particles (Sigma-Aldrich), as described in previous studies [2 (link)]. All human cell lines were authenticated using STR (or SNP) profiling within the last three years and were mycoplasma-free cells.