Shcontrol plko 1puro shc002

The HNSCC cell lines HN12 (tumorigenic and metastatic), HN13 [42 (link)] and Cal27 (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified medium (DMEM, Sigma-Aldrich, Munich, Germany), supplemented with 10% fetal bovine serum (FBS, GIBCO, Carlsbad, CA, USA), antibiotics and antimycotics (Sigma-Aldrich) in a humidified atmosphere of 5% CO₂ at 37°C. The MISSION short-hairpin RNA (shRNA) plasmid TCR1 containing DNA against human SET (shSET; TRCN0000063717; NM_003011.1-467s1c1; Sigma-Aldrich) or the shRNA control (shControl; pLKO.1puro; SHC002, shRNA non-mammalian target; Sigma-Aldrich) were transfected into HN12 cells using the Turbofect reagent (Thermo, Chelmsford, Massachusetts, USA). Stable transfectants were selected using puromycin (1 μg/mL). Lentivectors containing the shSET and shControl constructs were added to the HN13 and Cal27 cells for transduction. The transduced cells were selected using puromycin (1 μg/mL). For siRNA expression in the HNSCC cell lines, duplex RNA (siRNA) against SET was purchased from Qiagen; the protocol used for this experiment was previously reported [34 (link)]. The efficacy of SET knockdown was evaluated by Western blotting.