Hygromicin

Transfections were made by electroporation, performed using the Gene Pulser XCell^TM (BioRad, Hercules, CA, USA) by applying two sequential pulses of 1,500 V and 25 μF each as described previously^{72 (link),73 (link)}. The clonal selection was performed in semi-solid medium by addition of the antibiotics in the following concentrations: 600 µg/mL of Hygromicin (Invitrogen, Life Technologies), 80 µg/mL of Geneticin (G418) (Gibco/Thermo Fisher Scientific, Waltham, MA, USA) and 800 µg/mL Zeocin (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA). Mutant parasites were maintained in the presence of specific antibiotics for the resistance markers acquired at transfection. KH1 gene deletion was confirmed by PCR, real-time qPCR and Southern blot. Conventional PCR was applied to verify the correct integration of KH1 knockout cassette by using a reverse primer on HYG or NEO sequences and an external forward primer outside the cassette (Supplementary Fig. S1). Quantitative PCR was performed using genomic DNA to measure KH1 gene copy number and Southern blot directly detected KH1 by DNA probing hybridization.

MCA-203 fibrosarcoma cell line was maintained in DMEM (Lonza) supplemented with 10% heat-inactivated FBS (Gibco), 2 mM L-glutamine (Gibco), 10 mM HEPES (Lonza), 55 uM 2-mercaptoethanol (Gibco), 150 U/ml streptomycin, 200 U/ml penicillin obtained from the American Type Culture Collection (ATCC). Two additional cell lines were generated by the stable transfection with either electroporation or the usage of Lipofectamine 2000 (Invitrogen) of MCA cell line with the plasmid encoding either GM-CSF or its mutant version GM-CSF W30L. Transfected cells were selected and thus culture in the media used for MCA-203 supplemented with 0,3 ug/ml Hygromicin (Invitrogen).