Hcc1937

Human breast carcinoma cell lines HCC1937 and HCC1143, pancreas adenocarcinoma cell line PA-TU-8902, prostate carcinoma cell line DU145 and lung carcinoma cell line NCI-H460 were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany).
HCC1937, HCC1143, DU145 and NCI-H460 cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 1% Penicillin – Streptomycin (Sigma-Aldrich). PA-TU-8902 cells were cultured in Dulbecco’s MEM High Glucose (Sigma-Aldrich) supplemented with 2 mM L-Glutamine, 1 mM Sodium Pyruvate, 10% fetal calf serum and 1% Penicillin – Streptomycin in a humidified atmosphere with 5% CO₂ at 37°C. Cell lines were maintained in exponential growth and cells from subconfluent monolayers were harvested by trypsin-EDTA (Sigma-Aldrich) to carry out the experiments. For measurement of the parameters, the cell cultures were used within 4–6 weeks after thawing.

MDA-MB-231, BT-549, BT-20, HCC1937, MCF-7, T-47D, ZR-75-1, SK-BR-3 cell lines were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). All cell lines were grown in monolayers in appropriate media supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Sigma-Aldrich): RPMI 1640 Medium for BT-549, T-47D, HCC1937 and ZR-75-1 cells; Minimum Essential Medium for MCF-7 and BT-20 cells; McCoy’s 5A Medium for SK-BR-3 cells; Leibovitz’s L-15 Medium for MDA-MB-231 cells. All cells were maintained at 37°C in humid air with 5% CO₂ condition (BT-549, T-47D, HCC1937, ZR-75-1, MCF-7, BT-20 and SK-BR-3), or without CO₂ (MDA-MB-231). All cell lines were authenticated by STR analysis.

ChIP assays were performed using the BLBC cell lines HCC1937 and HCC1806 following the manufacturer's instructions (Abcam, Cambridge, MA, USA). Protein A/G beads (MCE, HY-K0202-1) were first mixed with an equal amount of anti-KLF5 antibody (R&D system, AF3758) or rabbit IgG (Proteintech) and incubated overnight at 4°C. HCC1937 and HCC1806 cells were fixed with 1% formaldehyde (Sigma) for 15 min at room temperature (RT). Glycine (125 mM) was added to quench the formaldehyde and terminate the cross-linking reaction. The cells were scraped into an Eppendorf (EP) tube and centrifuged at 500×g at 4°C for 10 min. The supernatant was aspirated off, and the pellet was resuspended in cytoplasmic lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% Nonidet P-40 with protease inhibitors; 1000 μL per 1×10⁷ cells). The cell suspension was centrifuged at 4000×g for 5 min at 4°C. Finally, the pellet was resuspended in nuclear lysis buffer (50 mM Tris-HCl pH 8.1, 10 mM EDTA, 1% SDS with protease inhibitors; 500 μL per 1×10⁷ cells) and then sonicated for ten cycles, with 30 s on and 30 s off for each cycle. The DNA‒protein complex was mixed with the antibody-A/G bead complex and incubated at 4°C overnight. The chromosomal DNA was purified by phenol/chloroform extraction and ethanol precipitation. The pellets were dissolved in 100 μL of ddH₂O for qPCR. Primers are listed in Supplementary Table 1.

The human breast cancer cell line JIMT-1 (ACC589) was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ) and was routinely maintained in Dulbecco's modified Eagle's medium/nutrient mixture Ham's F12 medium (VWR, Lund, Sweden). The human breast cancer cell lines MCF-7 (HTB-22), HCC1937, and human normal-like breast epithelial cell line MCF-10A (CRL-10317) were obtained from American Type Culture Collection (Manassas, VA, USA) and were cultured in RPMI1640 medium (VWR). The JIMT-1, MCF-7, and HCC1937 cell lines were cultured with the addition of 10% fetal calf serum (FCS) (VWR), nonessential amino acids (1 mM) (VWR), insulin (10 μg mL⁻¹) (Sigma-Aldrich), penicillin (100 U mL⁻¹) (VWR), and streptomycin (100 μg mL⁻¹) (VWR). In addition, HCC1937 cell line was also supplemented with epidermal growth factor (20 ng mL⁻¹) (Sigma-Aldrich). The MCF-10A cells were cultured with the addition of 10% heat-inactivated FCS, nonessential amino acids (1 mM), insulin (10 μg mL⁻¹), penicillin (100 U mL⁻¹), streptomycin (100 μg mL⁻¹), epithermal growth factor (20 ng mL⁻¹), cholera toxin (50 ng mL⁻¹) (Sigma-Aldrich), and hydrocortisol (250 ng mL⁻¹) (Sigma-Aldrich). All cell lines were maintained at 37 °C in a humidified incubator with 5% CO₂.

MCF10A MI and MII cells were obtained from Dr Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, USA) and maintained at 37 °C and 5% CO₂ in DMEM/F12 (Gibco), supplemented with 5% fetal bovine serum (FBS) (Biowest), 20 ng/ml EGF (PeproTech), 100 ng/ml cholera toxin (Sigma-Aldrich), 0.5 µg/ml hydrocortisone (Sigma-Aldrich), 10 µg/ml insulin (Sigma-Aldrich) (complete medium). Cells were starved in DMEM/F12 supplemented with 0.2% FBS, 100 ng/ml cholera toxin, 0.5 µg/ml hydrocortisone, and 10 µg/ml insulin, with or without 20 ng/ml EGF for 16 h prior to TGFβ treatment. HCC1954 breast cancer cells (obtained from Dr Andrew J. G. Simpson, Ludwig Cancer Research, New Your, USA), HCC1937 and HCC202 breast cancer cells (obtained from SE Le Dévédec, Leiden Academic Center for Drug Research, Leiden, the Netherlands) were maintained in RPMI-1640 (Sigma-Aldrich), supplemented with 10% FBS (Biowest). Cells were kept in 0.2% FBS starvation media with or without EGF (20 ng/ml) for 16 h prior to TGFβ treatment. The cell lines were frequently tested for absence of mycoplasma and were authenticated by identity testing.
A detailed description of the materials and methods, including the primer sequences used for qRT-PCR (Table S1) and ChIP-qPCR (Table S2), used in this study is available in the online Supplementary Material and Methods.