Cell line nucleofector kit r

For stable transfection experiments in ESCs, the sequences coding for E2F6 WT, as well as E68 and MB4 mutants, were inserted into the pCAG-EGFP-IB vector^{60 (link)}. To generate the E68 DNA-binding mutant, the amino acids in positions 68 and 69 were changed from Leu-Val to Glu-Ser^{18 (link)}. To generate the MB4 mutant, the E2F6 marked box domain (aa 180–240) was replaced by the marked box domain of E2F4 (aa 138–198). These constructs were transfected into ESCs by electroporation with the Amaxa Nucleofector II device using the Amaxa Cell Line Nucleofector kit R and stable transfectants were selected with blasticidin (7.5 μg/ml). For the DNA-binding domain swap experiment, the sequences coding for E2F6 WT, DBD1, and MB4 mutants were inserted in the lentiviral expression vector pCDH-MSCV-MCS-EF1α-GFP + Puro (System Biosciences). The DBD1 mutant was generated by replacing the DNA-binding domain of E2F6 (aa 61–129) with the DNA-binding domain of E2F1 (aa 122–187). Viral particles were produced in HEK293T cells and ESCs were infected with 8 μg/ml polybrene. Twenty-four hours after transduction, puromycin (2 μg/ml) was added for 72 h before collecting the RNA samples.

HeLa (DSMZ) human cervical cancer cells were propagated in DMEM (Dulbecco’s Modified Eagle Medium; PAA) supplemented with 10% fetal bovine serum (PAA), penicillin (PAA) and streptomycin (PAA) at 37°C in 5% CO2. For BAC DNA transfection 1 μg of BAC DNA purified with Large-Construct Kit (Qiagen) was mixed with 5 × 10⁶ HeLa cells and transfection was performed using Amaxa Nucleofector program A-28 and Cell Line Nucleofector Kit R (Amaxa). 72 hours later 400 μg/ml G418 (Sigma) was added to the growth medium to select for BAC containing cells. After two months of selection, single EGFP-positive clones were isolated with FACSAria cell sorting system (Becton-Dickinson). The cell lines were routinely grown in medium containing 800 μg/ml G418. For transfection of plasmid constructs cells were seeded into white 96-well clear bottom microtiter plate (Greiner Bio-One) at 10 000 cells per well in a volume of 200 μl of G418-containing culture medium. The next day medium was replaced with 100 μl of medium without G418 and cells were transfected with GenJet Hela transfection reagent (SignaGen Laboratories) using 100 ng of DNA per well at 1:3 DNA to lipid ratio. NPAS4 and ARNT2 constructs were cotransfected at 1:1 ratio. Five hours post-transfection medium was replaced with 200 μl of G418-containing medium.

COS-7 cells were transiently transfected with 5 µg of the indicated plasmid using the Amaxa Cell Line Nucleofector Kit R according to the manufacturer’s protocol. To generate soluble Fc fusion proteins of each vCD48, CD48, and CD58, HEK-293T cells were transiently transfected with 0.2 µg/cm² of the indicated plasmid mixed with 6 µL/µg DNA of polyethylenimine (1 mg/mL, Sigma-Aldrich) in 0.1 mL/cm² of OPTIMEM medium (Gibco, Thermo Fisher Scientific) for four hours. Then, cultures were washed and 6 days later the supernatants containing the Fc fusion proteins were collected, clarified to remove cellular debris, and concentrated 20-fold using the Amicon Ultra-15 Centrifugal Filter Unit with an Ultracel-30 membrane (Merck Millipore). The quantification of Fc fusion proteins was performed by sandwich ELISA employing anti-human Fc IgG mAb (clone 29.5; Fc specific; [22 (link)]) and anti-human IgG (Fc specific; Sigma-Aldrich) peroxidase (POD).