Du4475

Human breast cancer cell lines, MCF7, T47D, ZR75, MDA-MB-231, MDA-MB-468, SKBR3, Hs578T and HEK293T, DU4475 and MDA-MB-157 were purchased from ATCC, USA. Human 4-OH tamoxifen resistant breast cancer cells (LCC2) were obtained from Prof. Robert Clarke. RKO, RKO-T29, RKO-A19, HCT116, HCT116-HWT, HCT116-HMUT were obtained from Dr. Jihye Yun and Dr. Bert Vogelstein and culture methods were performed as described previously (Yun et al., 2009 (link)). MCF7, MDA-MB-231, MDA-MB-468, MDA-MB-157, Hs578T, HEK293T were grown in DMEM (Invitrogen), supplemented with 10% fetal calf serum (FCS) (Invitrogen), 100 U/ml penicillin/streptomycin and 1% sodium pyruvate. ZR75, T47D, SKBR3 and DU4475 were grown in RPMI, supplemented with 10% FCS, 100 U/ml penicillin/streptomycin and 2 mM L-Glutamine (ThermoFisher Scientific). LCC2 were grown in phenol red-free DMEM, supplemented with 10% charcoal stripped serum (CCS; ThermoFisher Scientific), 100 U/ml penicillin/streptomycin and 1% sodium pyruvate. All cell lines were grown at 37°C with 5% CO₂ in a humidified environment. Mycoplasma detection of all cell lines were tested and negative results were observed. Authenticated cell lines were used within six months of purchase from the ATCC.

The DU4475 and MDA-MB-231 human breast adenocarcinoma cell lines, and the HT29 and COLO205 colorectal adenocarcinoma cell lines were obtained from ATCC (Manassas, MD, USA). All cell lines were cultured in RPMI/10% FCS and maintained at 37 °C in 10% CO₂.
The murine parental IgG2a 2H10 antibody and the IgG1 reformatted 2H10 antibody, recognizing human and murine VEGF-B isoforms, and the IgG2a isotype control, were provided by CSL Ltd. (Parkville, VIC, Australia). Commercially available antibodies were obtained for human VEGFR-1 (rabbit polyclonal sc-9029 from Santa Cruz, Dallas, TX, USA, and 1527-PABX from Thermo Scientific, Scoresby, VIC, Australia) and VEGFR-2 (rabbit polyclonal sc-504, Santa Cruz, Dallas, TX, USA,; rabbit monoclonal antibody 55B11, Cell Signaling, Danvers, MA, USA). Secondary antibodies were FITC-labeled goat anti-rabbit IgG polyclonal antibody (ABR Affinity Bioreagents, Golden, CO, USA), and FITC-labeled goat anti-mouse IgG polyclonal (Calbiochem/Merck, Bayswater, VIC, Australia). Avastin (bevacizumab), a humanized anti-VEGF-A antibody (Roche/Genentech, San Francisco, CA, USA), specific for all human but not murine or rat VEGF-A isoforms [36 (link)], was provided by CSL Ltd.

Human breast cell lines HCC1143, DU4475, MDA-MB-468, BT-474, MDA-MB-134-VI, AU565 and MDA-MB-453 and T47D, were purchased from ATCC (Manassas, VA). CAL-51 and CAL-120 cell lines were a kind gift from Dr. Toru Ouchi (Roswell Park Cancer Institute, NY). Cells were grown in DMEM medium (Corning Cellgro, NY), supplemented with 20% fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 µg/ml) under the 5% CO₂ culture condition at 37 °C. VGLL4-WT was PCR amplified and inserted into the pBABE retroviral expression vector. VGLL4-ΔTUD1, ΔTUD2, and ΔTUD1&2 expression vectors were kindly provided by Dr. Ji^{4 (link)}.

The human TNBC cell lines, DU-4475 and MDA-MB-231 were purchased from ATCC (Manassas, VA, USA). ATCC authenticated them by short tandem repeat profiling, cell morphology monitoring, karyotyping, and cytochrome C oxidase I testing. The cells were grown in RPMI-1640 and DMEM media, respectively, with 10% FBS and 1% penicillin-streptomycin (Thermo Fisher Scientific, Pittsburgh, PA, USA). They were cultured at 37 °C in humid atmosphere containing 5% CO₂. Kinase inhibitors were purchased from Selleckchem (Munich, Germany), LC Laboratories (Woburn, MA, USA), MedChemExpress (Monmouth Junction, NJ, USA), or AdooQ Bioscience (Irvine, CA, USA).

Human cancer cell lines MA-11, MDA-MB-231 (ATCC #HTB-26™), Hs578T (ATCC #HTB-126™), AU565 (ATCC #CRL-2351™), HCC1428 (ATCC #CRL-2327™), MCF7 (ATCC #HTB-22™), HCC1187 (ATCC #CRL-2322™), DU4475 (ATCC #HTB-123™), HCC1569 (ATCC #CRL-2330™), DLD1 (ATCC #CCL-221™), HPAF-II (ATCC #CRL-1997™) and LNCaP (ATCC #CRL-1740™), were maintained at 37°C in 5% CO₂ and cultured in either Dulbecco´s Modified Eagle's Medium (DMEM) or Roswell Park Memorial Institute Medium (RPMI) supplemented with 10% FBS.
All cancer cell lines, except MA-11, were obtained from the American Type Culture Collection (ATCC; www.atcc.org). The human MA-11 breast carcinoma cell line, established from bone marrow micrometastases of a patient with breast cancer [52 (link)], was obtained by Øystein Fodstad (Norwegian Radium Hospital, Oslo, Norway). Cell pellets and conditioned media from the Melmet 1 (MM1) and Melmet 5 (MM5) melanoma cell lines were a gift from Dr. Siri Tveito at the Norwegian Radium Hospital, Oslo, Norway, and were established from the biopsies of metastatic melanoma patients at the department of Tumor Biology, The Norwegian Radium Hospital Oslo, Norway [53 (link)].

From ATCC (Manassas, VA, USA), human breast epithelial cell (MCF-10A), and BC cell lines (MCF-7, L6, MDA-MB-231, and DU4475) were bought. MCF10CA1a cell line was obtained from Karmanos Cancer Institute (Detroit, MI, USA). Dulbecco’s modified Eagle medium (DMEM, Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum, 100 μg/ml streptomycin, and 100 U/ml penicillin (Invitrogen, Carlsbad, CA, USA) was used for culturing the cells mentioned above in 5% CO₂ and 95% humidity at 37°C. The medium was refreshed every 48 h, and the cells were sub-cultured every 96 h.

PC-3, 22Rv1, LNCaP, VCaP, DU145, MDA-MB-231, ZR-75-1, BxPC3, 786-O, A498, HCT-116, HT-29, A2058, SkMel2, A375, OV90, PANC1, HCC1187, TCCSUP, RT4, HepG2, DU4475, HCC1806 and HCC1599 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cell lines derived from PC-3 (PC-3E and TEM 4-18^{9 (link)}; GS689Li and GS694Lad^{9 (link)}; TEM 4-18 ZEB1 KO) were previously generated in our laboratory. The identity of PC-3E, TEM 4-18, GS689Li and GS689Lad as PC-3 derivatives wes confirmed by STR profiling (IDEX Laboratories). All cell lines were grown at 37 °C in a 5% CO₂ atmosphere in ATCC-specified medium containing 10% fetal bovine serum and 1% non-essential amino acids. Monensin (M5273, 90–95% purity), salinomycin (S4526), nigericin (N7143), pyrvinium pamoate (1592001), epirubicin hydrochloride (E9406), gentian violet (48770), dactinomycin (1162400) and daunorubicin (D8809) were purchased from Sigma (Saint-Louis, MO).