Mda mb 468

Human breast cancer cell lines MDA-MB-231, MCF-7, BT-549, BT-20, MDA-MB-468 and HCC-1806 were obtained from American Type Culture Collection (ATTC, LGC Standards, London, UK). MDA-MB-231 and MCF-7 cell lines were cultured in DMEM/F-12 medium, BT-549, BT-20 and HCC-1806 cells in RPMI 1640 medium and MDA-MB-468 in DMEM high glucose medium (all from Gibco, Thermo Fisher Scientific, Madrid, Spain). All media were supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Madrid, Spain), 1% antibiotic-antimycotic mixture, 2 mM L-glutamine, 1× non-essential amino acids and 1 mM of sodium pyruvate (all from Gibco, Thermo Fisher Scientific, Madrid, Spain). MDA-MB-231-, HCC1806- and MDA-MB-468-ALDH1A1:tdTomato CSC models, in which CSC can be identified by the expression of tdTomato fluorochrome [13 (link)], were cultured in complete medium supplemented with the selection antibiotic blasticidin at 1 μg/mL (Gibco, Thermo Fisher Scientific, Madrid, Spain). Of note, the stemness nature of tdTomato-expressing cells from all three-CSC models has already been fully confirmed in vitro by increased expression of stemness markers, mammosphere formation and in vivo tumorigenic capacity [13 (link)]. All cell lines were incubated in a humidified incubator at 37 °C containing 5% CO₂.

Human normal breast cell line MCF‐10A and BC cell lines MCF‐7 and MDA‐MB‐468 were acquired from American Type Culture Collection (Manassas, VA, USA). MCF‐10A cells were cultured in MEBM BulletKit (Lonza, Basel, Switzerland). MCF‐7 and MDA‐MB‐468 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Sigma, Louis, Missouri, MO, USA) supplemented with fetal bovine serum (FBS, 10%, Sigma), streptomycin (100 μg/mL, Sigma), and penicillin (100 U/mL, Sigma). These cells were kept in a moist atmosphere with 5% CO₂ at 37°C.
Small interference (si) RNA targeting circ_0000518 (si‐circ_0000518) and corresponding control (si‐NC), as well as miR‐326 mimic and inhibitor (miR‐326 and anti‐miR‐326) and their matching negative controls (miR‐NC and anti‐miR‐NC), were obtained from GenePharma (Shanghai, China). The overexpression vectors of FGFR1 (pcDNA‐FGFR1) and circ_0000518 (circ_0000518) were established via cloning the full‐length sequence of FGFR1 or circ_0000518 into the pcDNA3.1 vector (pcDNA‐NC) (Invitrogen, Carlsbad, CA, USA) or pcD‐ciR (Geneseed Biotech Co., Ltd., Guangzhou, China) (Vector). BC cells (MCF‐7 and MDA‐MB‐468) were transfected with oligonucleotides or vectors using Lipofectamine 3000 reagent (Life Technologies, Grand Island, NY, USA).

Cell lines (TNBC, MDA-MB-468; non-TNBC, MCF-7) were provided by the Shanghai Type Culture Collection of the Chinese Academy of Sciences. MDA-MB-468 cells were cultivated with modified L-15 medium (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin–streptomycin at 37 °C under a humidified atmosphere with 0% CO₂. MCF-7 cells were cultivated with DMEM medium (10% FBS + 1% penicillin–streptomycin; 37 °C, 5% CO₂).