Plko 1 puro lentiviral vector

Commercial breast cancer cell lines MCF-7, T-47D, ZR-75–1, SK-BR3, MDA-MB-231, MDA-MB- 436, BT-549, HBL-100 and the non-tumorigenic breast cell line MCF-10A were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum and 1% penicillin– streptomycin (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) at 37 °C and 5% CO2. Cells with the doxycycline (DOX)-inducible ZEB1 expression, MCF-7/ZEB1^{40 (link)}, were maintained in the presence of absence of 1 μg/ml DOX for 72 h prior the experiments were carried out. To generate MDA-231 and Hs 578-T cells with the stable ZEB1 knockdown, cells were infected with the pLKO.1- PURO lentiviral vectors (Sigma-Aldrich, St. Louis, MO, USA) expressing ZEB1-targeting short hairpin RNA (shRNA) or control shRNA. Selection of cells expressing shRNAs was performed in 0.5 μg/ml puromycin-containing DMEM for 7–10 days. For transient ZEB1 depletion, short interfering RNA (siRNA) was purchased from Thermo Fisher Scientific (Waltham, MA, USA) (siRNA#1, and #2, Catalogue numbers 229970 and 229,971). siRNA transfection was performed using LTX Lipofectamine with Plus reagent (Thermo Fisher Scientific).

pLKO.1-puro lentiviral vectors expressing SOX2 shRNA and control shRNA (SHC002) were purchased from Sigma-Aldrich. For generation of lentiviral particles, HEK293FT cells were co-transfected with the shRNA lentiviral plasmid (pLKO.1-puro) and ViraPower Lentiviral packaging mix (pLP1, pLP2, pLP-VSV-G; Life Technologies) using Lipofectamine 2000 and the culture supernatants containing lentivirus were harvested at 48 h after transfection. For lentiviral transduction, cells were treated with the shRNA-expressing lentivirus in the presence of 5 μg/mL polybrene (Sigma-Aldrich, MO). To ensure shRNA-mediated silencing of SOX2 expression, the mRNA levels of SOX2 and GAPDH were determined by RT-PCR analysis.
Retroviral plasmids carrying the pMX-SOX2 were kindly provided by Jeong Beom Kim (UNIST, Republic of Korea) and individually co-transfected with packaging plasmids (gag-pol and VSV-G) into Plat-GP cells using Lipofectamine-Plus reagent. Plat-GP cells (RV-103, Cell Biolabs, Inc., San Diego, CA) were maintained in DMEM with high glucose, 10% FBS, 10 μg/mL blasticidin, and 1× penicillin-streptomycin. Virus-containing supernatants were collected two days after transfection, passed through a 0.45 μm filter, and stored at −80°C.

MEFs and C2C12 cells were grown in DMEM media supplemented with 10% FBS and 1% antibiotics. Primary myoblasts isolated from mouse hind limb muscles were grown in DMEM/F10 media supplemented with 20% FBS, 1% antibiotics, and 10 ng/mL bFGF. Cdk4, E2F3, or p16 knockdown in C2C12 cells or primary myoblasts was accomplished using shRNA in pLKO.1-puro lentiviral vectors (Sigma-Aldrich). pCMV-E2F3 plasmid or adenovirus Ad-human CDK4 (Vector Biolabs) was used for ectopic overexpression in C2C12 cells. Myogenic differentiation of C2C12 or primary myoblasts was initiated by switching the cells to medium containing 2% or 5% horse serum and continued for 4–6 days, until myotubes were visible. Cdk4 inhibitor (100 nM, IDCX), was included in myocyte differentiation media during the entire differentiation period.