Stable CIITA Overexpression in DFT Cells

DFT1 and DFT2 cell line C5065 and JV, respectively, were transfected with plasmid pCO2 to generate stable cell lines that overexpress CIITA. DNA transfections were performed using polyethylenimine (PEI) (1 mg ml⁻¹, linear, 25 kDa; Polysciences, Warrington, FL, USA) at a 3 : 1 ratio of PEI to DNA (w/w) as previously described [8 (link)]. Briefly, DFT cells were co-transfected with pCO2 and SB transposase vector pCMV(CAT)T7-SB100 [33 (link)] (a gift from Zsuzsanna Izsvak; Addgene plasmid no. 34879) at a ratio of 3 : 1 in µg, respectively. One microgram of total plasmid DNA was used per millilitre of culture volume. The cells were incubated with the transfection solution overnight at 35°C with 5% CO₂. The media was removed and replaced with fresh complete RPMI medium. Forty-eight hours of post-transfection, the cells were observed for expression of reporter gene mTagBFP. Positively transfected cells were selected with 1 mg ml⁻¹ hygromycin B (Sigma-Aldrich) for 7 days before being maintained in 200 µg ml⁻¹ hygromycin B in complete RPMI medium. The two tumour cell lines were also transfected with empty vector pSBbi-BH as controls.