Foetal calf serum

HT-29 (HTB-38™) and Caco-2 (HTB-37™) human colorectal cancer cell lines obtained from ATCC, were cultured in low glucose Dulbecco’s Modified Eagle’s Medium (DMEM) which contained 5% heat-inactivated foetal calf serum. Culture medium and foetal calf serum were obtained from Highveld Biologicals, South Africa. Cells were grown in 75 cm² culture flasks and incubated at 37°C in a humidified atmosphere with 5% CO₂. Both cell lines were subcultured when they reached confluence. Briefly, cells were washed twice with 10 ml phosphate buffered saline followed by the addition of 4 ml trypsin and incubation at 37°C for 10 minutes. Detached cells were reseeded to the same flask. Camptothecin (Sigma), a known inducer of apoptosis, and an inhibitor of topoisomerase-1, was used as a positive control in all experimental procedures.

Human cervix adenocarcinoma HeLa cells (ATCC; CCL-2; Manassas, VA, USA) and 293T cells (ATCC; CRL-3216) were grown in Dulbecco’s modified Eagle’s Medium (DMEM; Life Technologies, Darmstadt, Germany), with high glucose, supplemented with 10% foetal calf serum (Life Technologies), 100 U/ml penicillin and 100 μg/ml streptomycin (both Life Technologies) at 37 °C and 8% CO₂. The cells were regularly tested for mycoplasma contamination by a PCR-based assay. Jurkat cells (clone E6.1; ATCC; TIB-152) were maintained at 37 °C and 5% CO₂ in suspension in RPMI medium supplemented with 10% foetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin. A transient knockdown of flotillins in HeLa cells was performed as described earlier^{19 (link), 51 (link), 56 (link)} using the Stealth siRNA System (Life Technologies). GGA3 siRNAs were purchased from Life Technologies. For control transfections, an oligo that does not target any human sequence (Stealth RNAi Negative Control, medium GC content) was used. Experiments were performed 72 h after transfection. For growth factor stimulation, the cells were kept under serum-free conditions for at least 18 h before the experiment. Stimulation with EGF was performed with a final concentration of 100 ng/ml and 37 °C for the indicated time points.