The BPLER and HMLER cells were established previously20 (link) and characterized extensively4 (link),20 (link),90 (link)–98 (link). The BPLER cells are cultured in the BMI-T medium (US Biological, cat# 506387.500, or TumoriGenesis Product No: 833)5 (link), and the HMLER cells are cultured in MEGM medium (Lonza,cat# CC-3150)4 (link),20 (link). The BPLER and HMLER cells we established were tested for mycoplasma and deposited to ATCC (American Type Culture Collection, ATCC item #s CRL-3546 and CRL-3547) and European Collection of Authenticated Cell Cultures (ECACC, Accession numbers; 20012030, 20012033, 20012038, 20012041, 20012044, and 20012047, https://www.ukbrcn.org/news/new-accessions-coming-soon-to-the-european-collection-of-authenticated-cell-cultures/ ).
Megm medium
Manufactured by Lonza
Sourced in United States, Switzerland
MEGM medium is a serum-free, basal medium designed for the growth and maintenance of human mammary epithelial cells (HMECs). It provides the essential nutrients and growth factors required for the optimal proliferation and differentiation of HMECs in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Am4611, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by American Type Culture Collection (2 mentions)
Am16708, by Thermo Fisher Scientific (2 mentions)
F 12k medium, by American Type Culture Collection (2 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (2 mentions)
Mycoalert mycoplasma detection kit, by Lonza (2 mentions)
Dulbecco s modified eagle s medium, by Corning (2 mentions)
Mcf 10a, by Lonza (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Standard plates, by BD (2 mentions)
Primaria plates, by BD (2 mentions)
Mcf 10a, by American Type Culture Collection (2 mentions)
Mcf 7, by American Type Culture Collection (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Ultra low attachment plate, by Corning (1 mentions)
Vp 16, by Merck Group (1 mentions)
Mcf 10a cell line, by American Type Culture Collection (1 mentions)
22 protocols using megm medium
1
Characterization and Validation of BPLER and HMLER Cell Lines
2
Culturing TNBC Cell Line Mammospheres
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The human TNBC cell lines, SUM159 and 2LMP, were cultured under adherent conditions, as previously described [56 (link)]. Nonadherent mammospheres (“CIC” conditions) were cultured with MEGM medium (Lonza, Walkersville, MD, USA) in ultra-low attachment plates (Corning Costar, Corning, NY, USA). After 3 days of culture in CIC conditions, SUM159 mammospheres grew as irregular clusters (60–120 μm) of tightly joined cells; 2LMP mammospheres grew as non-uniform aggregates (90–230 μm) of cells. Mammospheres from both cell lines maintained high viability (>95%) under these conditions. Cell culture and in vitro experiments were performed at 37 °C in a 5% CO2 humidified atmosphere unless specified otherwise.
3
Cell Culture and Genetic Manipulation Protocols
4
Cell Culture and Genetic Manipulation Protocols
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Human BT-549 TNBC, A549 (mutant KRAS) NSCLC, H460 (mutant KRAS) NSCLC and embryonic kidney HEK293 cells were cultured in RPMI1640 medium (ATCC, Manassas, VA, USA). MDA-MB-231 (mutant KRAS) TNBC cells were grown in Dulbecco’s modified Eagle’s medium (Corning, Manassas, VA, USA). PC-3 prostate cancer cells were grown in F-12K medium (ATCC). MCF-10A cells were cultured in MEGM medium (Lonza, Walkersville, MD, USA). Media were supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Cell authentication was performed by short tandem repeat analysis. Cells were monitored for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza, Rockland, MA, USA). Cells stably expressing a control scrambled shRNA (CshRNA), MUC1shRNA, ZEB1shRNA, DNMT3bshRNA, empty vector or MUC1-C were generated as described (30 (link)–32 (link)). Cells were transfected to express a control siRNA (AM4611; ThermoFisher Scientific, Waltham, MA, USA) or RASSF1A siRNA (AM16708; ThermoFisher Scientific) in the presence of Lipofectamine RNAimax reagent (Invitrogen, Carlsbad, CA, USA).
5
Cytotoxicity Evaluation of Isolated Compounds
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POD, VP-16, and TAX were purchased to Sigma-Aldrich (Sigma-Aldrich, Merck KGaA, Saint Louis, MO, USA). Rojas-Sepulveda, 2012 [21 (link)] isolated compounds 1 and 2. The MCF10A cell line (ATCC: CRL-10317) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in MEGM medium (Lonza group, Basil, Switzerland) in a 5% CO2 incubator at 37 °C. Cells were thawed according to the standard methods, and the experiments were performed using freshly thawed cells after three passages.
6
Breast Cancer Cell Line Characterization
7
Cell Culture and Treatment Methods
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HeLa, BT474 and MDA-MB231 cells were cultured in DMEM medium (Gibco, Schwerte, Germany) supplemented with 10% FCS (Gibco) and 0.2% Penicillin (100 U/ml)/streptomycin (100 μg/ml) (Gibco) at 37 °C in 5% CO2. NCI-H226 cells were cultured in RPMI (Gibco) medium supplemented with 10% FCS and antibiotics, while HMEC and HMEC-T cells were cultured in MEGM medium (Lonza, Cologne, Germany) supplemented with growth factors as recommended by the company. For treatment with EGF (Labgen, Germany), a concentration of 25 pg/μl was used. MG132 treatment (Calbiochem, Darmstadt, Germany) was performed 5 h before lysing of cells at a concentration of 10 μM. Cycloheximide chases were performed with InSolution Cycloheximide (Calbiochem) at a final concentration of 100 μg/ml.
8
Cultivation and Characterization of Human Cell Lines
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Normal human diploid foreskin fibroblasts (HDF), HDF expressing HPV16-E6 and human mammary epithelial cells (HMEC), from frozen stocks, were obtained, cultured and synchronized as described previously (Baus et al., 2003; Lossaint et al., 2011) . U2OS (human osteosarcoma) cells were originally purchased from ATCC (Manassas, VA, USA) and HCT-116 (colon carcinoma) cells were a gift of Dr. B.
Vogelstein (Johns Hopkins University, Baltimore, MD, USA) from 2000 to 2010. The human fibroblasts (BJ) expressing inducible SV40 mutant (T 121 ) specifically targeting pocket proteins (Conklin et al., 2012) were a gift from Dr. J. Sage (Stanford, CA, USA, Journal of Cell Science • Accepted manuscript in 2018). Cell lines were not authenticated but were weekly tested for mycoplasma contaminations (Mycolalert kit, Basel, Switzerland).
Excepting for HMEC (MEGM medium; Lonza, Basel, Switzerland) and HCT-116 (McCoy's 5A medium, Gibco® Life Technologies, Waltham, MA, USA) all cell lines were cultured in Dulbecco modified Eagle medium (DMEM, high glucose, pyruvate, GlutaMAX -Gibco® Life Technologies, Waltham, MA, USA) supplemented with 10% foetal bovine serum (Sigma-Aldrich, St Louis, MO, USA; Dutcher, Bernolsheim, France or HyClone, Cytiva, Marlborough, MA, USA). Cells were grown under standard conditions at 37°C in humidified incubator containing 5% CO 2 .
Vogelstein (Johns Hopkins University, Baltimore, MD, USA) from 2000 to 2010. The human fibroblasts (BJ) expressing inducible SV40 mutant (T 121 ) specifically targeting pocket proteins (Conklin et al., 2012) were a gift from Dr. J. Sage (Stanford, CA, USA, Journal of Cell Science • Accepted manuscript in 2018). Cell lines were not authenticated but were weekly tested for mycoplasma contaminations (Mycolalert kit, Basel, Switzerland).
Excepting for HMEC (MEGM medium; Lonza, Basel, Switzerland) and HCT-116 (McCoy's 5A medium, Gibco® Life Technologies, Waltham, MA, USA) all cell lines were cultured in Dulbecco modified Eagle medium (DMEM, high glucose, pyruvate, GlutaMAX -Gibco® Life Technologies, Waltham, MA, USA) supplemented with 10% foetal bovine serum (Sigma-Aldrich, St Louis, MO, USA; Dutcher, Bernolsheim, France or HyClone, Cytiva, Marlborough, MA, USA). Cells were grown under standard conditions at 37°C in humidified incubator containing 5% CO 2 .
9
Mammosphere Formation and Quantification
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Cells were seeded on ultra-low attachment plates [42 (link)] at a density of 200 cells/200 μl/well in MEGM medium (Lonza) supplemented with B27, 10 ng/ml bFGF, 20 ng/ml EGF. After incubating the cells for 4–5 d, the primary mammospheres were counted and visualized using a DMI 5000 Leica microscope. For secondary mammosphere formation assays, mammospheres were collected by centrifugation at 800 g for 5 min, resuspended in 100 μl of 0.05% trypsin and incubated at 37°C for 10 min, and further dissociated into single cells by pipetting, as verified by microscopy. Single cell dilutions were then replated on ultralow attachment plates, and colonies were quantified as for the primary mammosphere assays.
10
Cell Line Propagation and Fingerprinting
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