Transcriptional responses to TGFβ1 and conditioned media in mammary epithelial cells
Transcriptional responses to TGFβ1 and conditioned media in mammary epithelial cells
Non-tumorigenic human breast epithelial MCF10A cells (authenticated in 2016) were cultured in DMEM/F12 medium supplemented with 5% horse serum (BioWest, Nuaillé, France), 5 µg/mL insulin (Sigma-Aldrich, Saint Louis, MO, USA), 0.5 µg/mL hydrocortisone (Sigma-Aldrich), 20 ng/mL EGF (Sigma-Aldrich). Breast cancer MCF7 cells were cultured in DMEM/F12 medium supplemented with 10% Fetal Bovine Serum (EURx, Gdansk, Poland). Cells were seeded on culture plates the day before the start with TGFβ1 stimulation, at various densities (~80% at the time of cells harvesting and RNA isolation). Each day TGFβ1 was administered in fresh medium for 2 h and then the medium was changed to fresh one for another 22 h to obtain conditioned medium (CM); CM from each treatment day was used to treat other cells to induce potential bystander effect. Total RNA isolation was performed at appropriate time points. Total RNA was isolated using the Direct-ZolTM RNA MiniPrep Kit (Zymo Research) and digested with DNase I (Worthington Biochemical Corporation). For each experimental point, RNAs from three biological replicates were first tested by RT-qPCR for the efficiency of treatments. Libraries were generated with Illumina TruSeq mRNA stranded (poly-A selection) with a customized fragmentation protocol (custom fragmentation protocol: 90'C for 2 minutes).