Mcf 7 cells

Adenocarcinomic human alveolar basal epithelial cells (A549 cells) and human breast cancer cells (MCF-7 cells) (ATCC) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO, USA), and were grown in an incubator with 5% CO₂ at 37°C.

MCF-7 and MDA-MB-231 cells were obtained from the American Type Culture Collection (ATCC, Boras, Sweden). Primary human fibroblasts were obtained, with approval of the local ethical committee (TMEK No. 2551), from skin biopsies using an in-house migration assay. MCF-7 cells were routinely maintained in EMEM (GIBCO, Grand Island, NY, USA), supplemented with 10% FBS (PAA, Pasching, Austria) and 10 μg/mL human insulin (Sigma, St. Louis, MO, USA). MDA-MB-231 and primary human fibroblasts were maintained in DMEM with high glycose (PAA), supplemented with 10% FBS (PAA). All media contained l-glutamine (0.1 mg/mL) (PAA) and were supplemented with 1% penicillin/streptomycin (PAA). Cells were cultured in a humidified atmosphere of 5% CO₂ at 37 °C.

MCF7 human breast cells were obtained from American Type Culture Collection (ATCC) in Manassas, VA, USA. Cells were cultured in 75 cm tissue culture flasks (Corning) in a humidified atmosphere (5% CO₂) at 37 °C. MCF7 cells were cultured in DMEM high glucose media (Gibco, Grand Island, NY, USA) with 10% FBS and penicillin–streptomycin (Gibco). Doxorubicin hydrochloride (DOX; Pfizer, Manhattan, NY, USA), tamoxifen (TAM; Cayman Chemical, Ann Arbor, MI, USA), APCIN (Sigma), Mad2-Inhibitor 1 (Cayman Chemical), nocodazole (Sigma), and thymidine (Sigma-aldrich cat # t1895) were acquired from the indicated providers. All treatment compounds were reconstituted in dimethylsulfoxide (DMSO). Drug treatments were applied at the concentrations and times as indicated. Flow cytometry was performed as described previously [32 (link)].

All cell lines were obtained from KeyGEN BioTECH (Nanjing, China). HeLa cells, A549 cells, MCF-7 cells, MDA-MB-231 cells and L02 cells were cultured in DMEM medium (Gibco, Grand Island, NY) containing 10% fetal calf serum (Gibco, Grand Island, NY), penicillin (100 μg/mL), and streptomycin (100 μg/mL) at 37 °C in a 5% CO₂ atmosphere. K562 cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY) containing 10% fetal calf serum (Gibco, Grand Island, NY), penicillin (100 μg/mL), and streptomycin (100 μg/mL) at 37 °C in a 5% CO₂ atmosphere. The cells were collected in the exponential phase of growth, and 1 million cells were dispersed in a 1.5-mL EP tube, washed twice with ice-cold phosphate-buffered saline (pH 7.4) solution and resuspended in 200 μL of ice-cold CHAPS lysis buffer (10 mM Tris-HCl, pH 7.5, 1 mM MgCl₂, 1 mM EGTA, 0.5% (W/V) CHAPS, 10% (V/V) glycerol). The lysates were incubated on ice for 30 min and then centrifuged at 12,000 rpm at 4 °C for 20 min. The upper cleared lysate solution was carefully transferred to a fresh RNase-free tube, flash frozen and stored at −80 °C before use.