Lipofectamine 8000

The human normal mammary epithelial cells MCF-10A and TNBC cell lines of BT-20, MDA-MB-468, and MDA-MB-231 (Procell) were grown at 37°C and 5% CO2 in RPMI1640 containing 10% fetal bovine serum (FBS, Gibco). The targeting KRT81 shRNA and negative control were synthesized by GenePharma. The sequences of sh-KRT81 and sh-NC were: 5′-ACCUGCGGAUCAGGAUUUGTT-3′ （sense）and 5′-CAAAUCCUGAUCCGCAGGUTT-3′ (anti-sense); 5′-UUCUCCGAACGUGUCACGUTT-3′ （sense）and 5′-ACGUGACACGUUCGGAGAATT-3′ (anti-sense), respectively. Lipofectamine 8000 (Beyotime) was used to transfect the siRNA constructs into the MDA-MB-231 cells. Cells were harvested for subsequent experiments 48 hours posttransfection. Full-length KRT81 DNA was cloned into the pcDNA3.1 (+) vector (GenePharma) and transfected to the BT-20 cell line by Lipofectamine 8000 for overexpression of KRT81.

B104 cells were cultured in high‐glucose DMEM medium (Cat. No. 10741574, Gibco, USA) supplemented with 10% fetal bovine serum (Cat. No. 11573397, Gibco, USA), and the cells were maintained in 5% CO₂ at 37°C. At 24 h prior to transfection assay, the cells were seeded into a 6‐well plate (4 × 10⁵ cells per well), a 12‐well plate (2 × 10⁵ cells per well), or a 96‐well plate (8 × 10³ cells per well) and allowed to grow to 80% confluence. The cells were transfected with pLV‐lncRNA INPP5F (VectorBuilder, China) and pLV‐NC (VectorBuilder, China). LncRNA INPP5F antisense oligonucleotide (ASO, RiboBio, China, Table S1), ASO NC (RiboBio, China; the sequence was protected by a patent from RiboBio), miR‐335 agomir (GenePharma, China, Table S1), agomir NC (GenePharma, China, Table S1), miR‐335 antagomir (GenePharma, China, Table S1), and antagomir NC (GenePharma, China, Table S1) were transfected into the B104 cells with lipofectamine 8000 (Cat. No. C0533, Beyotime, China). After 24, 48, or 72 h of transfection, the cells were used for subsequent experiments.