The human breast cancer cell lines SKBR3, AU565 and HCC1954 were kindly provided by Wirtz (Stratifyer GmbH, Köln, Germany). The cell lines T47D and MDA-MB-231, MDA-MB-231SA and MDA-MB-231-Brain were a generous gift from Professor Harriet Wikman–Kocher (Institute of Tumour Biology, University Medical Center Hamburg, Eppendorf, Hamburg) and Dr Takara (University of Texas), respectively. All cell lines were cultured under standard conditions in a water-saturated atmosphere containing 5% CO2 at 37 °C.
To generate cells with reduced MAN1A1 expression, MDA-MB-231 cells were transfected with five expression vectors containing different shRNA sequences targeting human MAN1A1 and a scramble negative control (shRNA nc; Origene, Herford, Germany) using Lipofectamin (Invitrogen GIBCO/Life Technologies, Carlsbad, CA, USA) as previously described (Oliveira-Ferrer et al, 2014 (link)). After puromycin (1 μg ml−1, PAA Laboratories GmbH, Pasching, Austria) selection, the level of MAN1A1 protein and mRNA was determined with western blot analysis and RT–qPCR, and two cultures (MAN1A1 shRNA #2 and MAN1A1 shRNA #3) were chosen for further analysis.
To generate cells with reduced MAN1A1 expression, MDA-MB-231 cells were transfected with five expression vectors containing different shRNA sequences targeting human MAN1A1 and a scramble negative control (shRNA nc; Origene, Herford, Germany) using Lipofectamin (Invitrogen GIBCO/Life Technologies, Carlsbad, CA, USA) as previously described (Oliveira-Ferrer et al, 2014 (link)). After puromycin (1 μg ml−1, PAA Laboratories GmbH, Pasching, Austria) selection, the level of MAN1A1 protein and mRNA was determined with western blot analysis and RT–qPCR, and two cultures (MAN1A1 shRNA #2 and MAN1A1 shRNA #3) were chosen for further analysis.
