Mebm bulletkit

UWB1.289 (UWB) and UWB1.289 + BRCA1 (UWB+B) cell lines were purchased from American Type Culture Collection (ATCC). Cells were incubated in 1:1 MEBM Bullet Kit and RPMI1640 media (Lonza) supplemented with 3% fetal bovine serum and maintained with 5% CO₂ at 37 °C. UWB+B cells were furthermore supplemented with 200 μg/ml G418 (Life Technologies, Inc). Cells were periodically tested for mycoplasma and authenticated using Short Tandem Repeat analysis.

Human TNBC cell lines (BT-549, MDA-MB-231, SUM-159, MDA-MB-453 and MDA-MB-468) and normal mammary epithelial cell line (MCF-10A) were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). 293 T cell lines were preserved by our lab. MDA-MB-231, MDA-MB-453, MDA-MB-468, SUM-159 and 293 T cells were cultured in DMEM (Gibco, Carlsbad, CA, USA), BT-549 cells were cultured with RPMI 1640 (Gibco, Carlsbad, CA, USA), containing 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin. MCF-10A cells were cultured in MEBM BulletKit (Lonza, Basel, Switzerland). All these cell lines were maintained at 37 °C with 5% CO ₂ in a humidified incubator.

The breast cancer cell lines MCF-7, MDA-MB-231, MDA-MB-468, T-47D, UACC-812, and MCF-10A were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). MCF-7, T-47D, MDA-MB-468 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; GIBCO, NY, USA) supplemented with 10% fetal bovine serum (FBS; HyClone). MCF-10A cells were cultured in MEBM BulletKit (Lonza, Basel, Switzerland). The two cell lines were cultured in a humidified incubator equilibrated at 37 °C in 5% CO₂. MDA-MB-231 and UACC-812 cells were cultured in L-15 (GIBCO) with 10% FBS without CO₂, supplemented with antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin; Sigma-Aldrich, USA). The cell lines in the experiments were validated by STR DNA analysis and were negative for mycoplasma.

HMLE cells were grown in a 1:1 ratio of DMEM:F12 (Thermo Fisher Scientific #11320-033) and MEBM + Bullet Kit (Lonza # CC-3150). T47D cells were gown in RPMI (Thermo Fisher Scientific #SH3002701) with 10% FBS (VWR #83007-198) and 1% Pen Strep (Thermo Fisher Scientific #10378-016).
We induced an EMT in HMLE or T47D cells by incubation with 2.5 ng/mL TGF-β, or ectopic expression of EMT-inducing transcription factors Snail, Slug, or Twist. Cells stably expressing these transcription factors, as well as ER-Snail, were a gift from the Sendurai Mani lab at The University of Texas MD Anderson Cancer Center. 4-Hydroxytamoxifen (Sigma-Aldrich #H7904) was added at a final concentration of 20 nM to ER inducible Snail HMLE cells.
We performed scratch-wound, transwell migration, and mammosphere formation assays as previously described [15 ].

Human BC cell lines (MDA-MB-231, MCF-7, SKBR-3, MDA-MB-453 and BT-549) and normal mammary epithelial cell line MCF-10A were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). MCF-7, MDA-MB-231 and MDA-MB-453 cells were cultured in DMEM medium (Gibco, Carlsbad, CA, USA), BT-549 and SKBR-3 cells were maintained in RPMI-1640 medium (Gibco), supplemented with 10% fetal bovine serum, 100 mg/mL streptomycin and 100 U/mL penicillin. MEBM BulletKit (Lonza, Basel, Switzerland) was used to culture MCF-10A cells. All these cells were maintained in a humidified incubator at 37 °C with 5% CO₂. As to hypoxic treatment, cells were cultured in a tri‑gas incubator with 1% O₂, 94% N₂ and 5% CO₂.

Human BC cell lines (MCF-7, MDA-MB-231, BT-549, MDA-MB-453, and SK-BR-3) and normal breast epithelial cell lines (MCF-10A) were available from the American Type Culture Collection (ATCC, Manassas, VA, USA). For these cells, MCF-7, MDA-MB-231, and MDA-MB-453 cells were cultivated in DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), BT-549, and SK-BR-3 cells were cultivated in the 10% FBS-contained RPMI-1640 medium (Gibco), and MCF-10A cells were cultured in MEBM Bullet Kit (Lonza, Basel, Switzerland). All these cells were subjected to cultivation in a humidified incubator at 37 °C with 5% CO₂.

The human cell lines MCF-10A, MDA-MB-231, and BT549 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), while MCF-7 and MDA-MB-453 cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). MDA-MB-231, MDA-MB-453, and MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) and BT549 cells were cultivated in Roswell Park Memorial Institute (RPMI)-1640 (Gibco) medium supplemented with 10% fetal bovine serum (FBS, Gibco). MCF-10A cells were cultured in MEBM BulletKit (Lonza, Basel, Switzerland). All cells were incubated in a humified 5% CO₂ incubator at 37 °C. Commercialized si-LINC01125, si-p53, and negative-control siRNA as well as pCDNA3.1-LINC01125 and pCDNA3.1-p53 plasmid were purchased from Thermo Fisher Scientific (MA, USA). MDA-MB-231 and BT549 cells were seeded in six-well plates at a density of 3 × 10⁵ cells/well overnight. Cell transfection was performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The sequences of siRNAs are presented in Supplementary Table S1. To knock down LINC01125, three siRNAs and a siRNA-NC were examined by qRT-PCR and siRNA-3 as the most effective one (Supplementary Figure S1A).