Mda mb 175

The estrogen receptor α‐positive MCF‐7 (ATCC HTB‐22), BT‐474 (ATCC HTB‐20), and MDA‐MB‐175 (ATCC HTB‐25) and the estrogen receptor α‐negative MDA‐MB‐231 (ATCC HTB‐26), BT‐549 (ATCC HTB‐122), and MDA‐MB‐468 (ATCC HTB‐132) human BrCa cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; HyClone, GE Healthcare Life Sciences, Pittsburgh, PA, USA) and 100 IU·mL⁻¹ penicillin and 100 IU·mL⁻¹ streptomycin (Life Technologies Australia Pty Ltd., Scoresby, Vic, Australia). Cell lines are validated periodically in house by Garvan Molecular Genetics using a test based on the Powerplex 18D Kit (DC1808, Promega, Madison, WI, USA) and tested for mycoplasma every 3 months (MycoAlert™ mycoplasma detection kit, Lonza, Basel, Switzerland).
To inhibit diacylglycerol O‐acyltransferase 1 (DGAT1) activity, cells were treated with AZD3988 (Tocris Bioscience, Invitrogen) (McCoull et al., 2012) or dimethyl sulfoxide (DMSO) control for 24 h in low‐glucose DMEM and no antibiotics. After treatment, cells were washed and sensitivity to palmitate‐induced apoptosis was assessed. To inhibit FA oxidation, cells were treated with etomoxir (Sigma‐Aldrich, Castle Hill, NSW, Australia) in low‐glucose DMEM and no antibiotics for times detailed in figure legends.

Human ER+/HER2-low breast cancer cell lines (MCF-7 [#HTB-22], T47D [#HTB-133], ZR-75-1 [#CRL-1500], and MDA-MB-175 [#HTB-25]) and HEK 293 [#CRL-1573] human embryonic kidney cells were obtained from the American Type Culture Collection. This supplier routinely authenticates cells lines by karyotyping, short tandem repeat profiling, assessment of cell morphology, and species verification by isoenzymology. Upon receipt, cell lines were expanded and aliquots frozen. Cells were passaged for a maximum of 6 months after resuscitation; after 6 months cells were discarded and new aliquots of cells were resuscitated. Cells were cultured in RPMI 1640 or MEM Eagle medium (PAN Biotech GmbH, Aidenbach, Germany) containing 10% fetal calf serum (FCS; PAA Laboratories GmbH, Pasching, Austria) at 37°C in a water-saturated atmosphere containing 5% CO₂.
The anti-HER3 antibody lumretuzumab, and the anti-HER2 antibodies pertuzumab (Perjeta^®) and trastuzumab (Herceptin^®) were obtained from Roche (Penzberg, Germany). The anti-estrogen fulvestrant (Faslodex^®) was obtained from AstraZeneca.

MDA-MB-175, MDA-MB-231, MDA-MB-453, MDA-MB-468, HCC-1143, HCC-1806, BT-20, BT-549, and HS-578T cells were from the American Type Culture Collection. All cells were cultured in a humidified incubator at 37 °C and 5% CO₂. MDA-MB-231, MDA-MB-453, HCC-1143, and HCC-1806 cells were cultured in RPMI-1640 + 10% FBS + 1% PS. BT-549 cells were cultured in RPMI-1640 + 10 µg/mL insulin + 10% FBS + 1% PS. HS-578T cells were cultured in DMEM + 10 µg/mL insulin + 10% FBS + 1% PS. BT-20 cells were cultured in DMEM + 1× amino acids + 10% FBS + 1% PS. MDA-MB-175 cells were cultured in DMEM + 10% FBS + 1% PS. MDA-MB-468 cells were cultured in DMEM/F12 + 10% FBS + 1% PS.

ER+ MCF7 (originally obtained from the Barbara A. Karmanos Cancer Institute, Detroit, MI, USA), MCF7(2) (a MCF7 derivative cell line that shows increased sensitivity to estrogen; [18 (link)] and TNBC cell lines, MDA-MB-231 and MDA-MB-468 (originally obtained from American Type Culture Collection, Manassas, VA, USA), were provided by the Tissue Culture Shared Resources at Georgetown University Medical Center. ER+ breast cancer cells line, MDA-MB-175, was purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). All cell lines were grown in IMEM (Life Technologies, Grand Island, NY, USA; A10489-01) media supplemented with 5% fetal bovine serum. BOLD-100 (formerly IT-139, NKP1339 and KP1339) was provided by Intezyne Biosciences, Inc. (Tampa, FL, USA). For in vitro assays, dimethyl sulfoxide (DMSO) was used at the diluent and negative control (0.2%). Olaparib (AZD-2281) was purchased from Selleck (Houston, TX, USA). All cells were authenticated by DNA fingerprinting and tested regularly for Mycoplasma infection. All other chemicals were purchased from Sigma-Aldrich.

The antibody specificity of anti-SLC7A5 (EPR17573, Abcam, UK) was validated using western blotting in human embryonic kidney (HEK) 293 T over expression lysate (Origene Technologies, Rockville, MD, USA) and MDA-MB-175 (luminal B-like), T47D and MCF7 (luminal A) [19 (link)] breast cancer lysate (American Type Culture Collection; Rockville, MD, USA). A dilution of 1:200 of the primary antibody and 1:1000 HRP-conjugated (Dako) secondary antibodies were applied: 5% milk /PBS-Tween (0.1%) (Marvel Original Dried Skimmed Milk, Premier Food Groups Ltd., UK) was used for blocking. Mouse monoclonal anti-β-actin primary antibody was used as a marker of endogenously expressed control. SLC7A5 bands were visualised using enhanced chemiluminescence (ECL) showing a single specific band at the correct predicted size (40 kDa) for the SLC7A5 protein.