24 well ultra low attachment plate

Cells were trypsinized, centrifuged and resuspended in cell medium without FBS. Each experimental group contained 2 × 10⁶ cells in 90 µL of cell medium without FBS and 10 µL of CDDP, OXA or BLM solution or 10 µL of cell medium without FBS in the case of untreated or electroporation only controls. The final concentrations of cytotoxic drugs were therefore 10 times lower than the working concentrations (CDDP: 40, 30, 20, 15, 10, 5, 2.5 µM; OXA: 80, 60, 40, 30, 20, 15, 5, 2.5 µM; BLM: 1.8, 1.3, 1, 0.5, 0.1, 0.01 nM). Then, 50 µL of the mixture containing 1 × 10⁶ cells was pipetted between two stainless steel plate electrodes (2.4 mm distance between the electrodes) and electroporated with 8 square wave pulses, 1300 V/cm, 100 µs duration at frequency of 1 Hz with electric pulse generator (Jouan GHT beta). The cell mixture was then transferred into the wells of a 24-well ultra-low attachment plate (Corning Costar), and 5 min after electric pulse delivery, 2 mL of cell culture medium was added. The other 50 µL of the mixture was not electroporated and only transferred into the wells of a 24-well ultra-low attachment plate (Corning Costar), and after 5 min 2 mL of cell culture medium was added.

Both the lung cancer cell line A549, purchased from ATCC, and head and neck cancer cell line UM-SCC 22A (SCC22A) [8 (link),37 (link)] were expanded and stored in liquid nitrogen. Cell lines were cultured in advanced DMEM (Gibco/Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 2 mM L-glutamine (Life Technology/Thermo Fisher Scientific, Inc., Waltham, MA, USA), Penicillin-Streptomycin (Gibco), plus 10% FBS. For three-dimensional (3D) sphere cultures, cells were plated in ultra-low attachment 24-well plates (Corning Costar, Corning, NY, USA) in serum-free medium containing TGFβ [5 ng/mL] or vehicle (1 mg/mL BSA in 4 mM HCl) and cultured for 6 days before analysis. All cell lines were tested for mycoplasma contamination by applying MycoSensor PCR Assay kit according to manufacturer’s instructions (Agilent Technologies, Inc., Santa Clara, CA, USA). For La-specific shRNA-mediated depletion lentiviral transduction of MISSION shRNA constructs (TRCN0000062193 and TRCN0000062195) were performed according to the manufacture’s instruction (Sigma-Aldrich, St. Louis, MO, USA). All lentiviral experiments were performed under biosafety level S2 at our laboratory at the Medical University of South Carolina (Charleston, SC, USA).