Illumina HiSeq 2500 sequencing; Estrogen receptor β role in tumour suppression of triple negative breast cancer
Estrogen receptor β role in tumour suppression of triple negative breast cancer
HCC1806 (CRL-2335), MDA-MB-468 (HTB-132) and Hs 578T (HTB-126) cell lines were purchased from the American Type Culture Collection (ATCC). Each cell line was grown in the appropriate culture media, according to manufacturer's protocol, and kept in an incubator at 37 ° C, in the presence of 5% CO2. Additional details are described in Supplementary Materials. Periodically, cells were tested for mycoplasma contamination using the PCR Mycoplasma detection kit (Applied Biological Materials). The selected cell lines were used to generate full-length ERβ-expressing experimental model, using the Clontech Lenti-X Tet-On Advanced Inducible Expression System. First, the 3xFlag-ERβ1 DNA fragment was cloned  into a pLVX-Tight-Puro vector, downstream to a tetracycline inducible promoter. The cloning procedure was performed using the Clontech In-Fusion HD Cloning Kit. Subsequently, to produce lentiviral particles expressing both rTA and Erβ1 genes,Clontech's Lenti-X Packaging Single Shots (VSV-G) were used according to manufacturer's protocol. Finally, the selected TNBC cell lines were infected first with the rtTA lentiviral particles, encoding for a a transcription factor that binds to a tightly regulated inducible promoter, after were transduced with 3xFlag-ERβ lentiviral particles and tested for the protein expression upon doxycycline (Sigma-Aldrich) induction Cell proliferation of ERβ-expressing clones was evaluated using the MTT (3-(4,5-Dimethylthiazol-2-yr)-2,5-Diphenyltetrazolium Bromide)-based spectrophotometric assay (M6494, Invitrogen), according to the manufacturer's instructions. Cells were plated on a 96-well plate and exposed to doxycycline or vehicle for 12 days in an incubator (37°C, 5% CO2). The amounts of doxycycline used to induce ERβ expression were the followings: 0.035 μg/mL of medium for HCC1806, 1.0 μg/mL medium for Hs 578T, 2.0 μg/mL medium for MDA-MB-468. Proliferation was evaluated at different time points: 0 days (starting point), 3, 6, 9 and 12 days (ending point), each experiment performed in different biological replicates and compared with vehicle-treated cells at same time points.  None Total RNA was extracted from HCC1806, MDA-MB-468 and Hs 578T ERβ clones, previously treated or not with doxycycline (0.035 µg/mL, 2.0 µg/mL and 1.0 µg/mL respectively) for 9 days, using TRIzol reagent (Life Technologies) according to the manufacturer's instructions As a control for doxycycline effects on total RNA expression of MDA-MB-468 cells, total RNA was extracted too from MDA-MB-468 RTA clones, previously treated for 9 days with doxycycline (2.0 µg/mL). Three independent biological replicates were performed for each cell line. Once extracted, RNA concentration and purity of each sample were assayed with NanoDrop™ 2000/2000c spectrophotometer (Thermo Fisher Scientific, USA) while its integrity was evaluated using TapeStation 2200 (Agilent, USA) instrument through RNA ScreenTape Assay
Experimental Factor: phenotype
not expressing ESR2