Bt 549

MCF-7, MDAMB-231, BT549, and SUM159PT cells were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). All carcinoma cell lines were cultured according to ATCC propagation instructions. By following the procedure in Basappa et al. [37 (link)], first, 2 × 10³ MCF-7 cells in 200 μL were grown in MEM enriched with 2% FBS and kept at 37 °C in a humidified 5% CO₂ environment. The compounds (10 mM) were dissolved in DMSO and were stored as a stock solution. The DMSO and the stock solution of compounds were diluted to 0.01, 0.1, 10, 100, and 1000 μM solutions in cell culture medium, keeping a DMSO amount less than 1%. MCF-7 cells (2 × 10³) were incubated for 72 h with exposure to pyrimidines and Alamar Blue reagent was used to evaluate cell viability.

Human breast cancer cell lines (MDA-MB-231, MDA-MB-468, BT549, MCF-7, and T-47D) were obtained from Procell (Wuhan, China). Cells were maintained in DMEM and RPMI 1640 supplemented with 10% FBS and 1% penicillin-streptomycin (100 U/mL penicillin and 100 mg/mL streptomycin) in an incubator with a 5% CO₂ humidified atmosphere at 37 °C.

Human breast cancer cell lines (MCF-7; BT-549; SKBR3; MDA-MB-231) and normal breast epithelial cell line (MCF-10A) were purchased from Procell (Wuhan, China). Cells were cultured in in DMEM containing 10% fetal bovine serum (FBS; C0400, VivaCell, Shanghai, China) and 1% penicillin/streptomycin (C0222, Beyotime, China) at 5% CO₂ and 37 °C. The DNAJB4 overexpression lentivirus and negative control were purchased from GeneChem (Shanghai, China). The DNAJB4 overexpression lentivirus and negative control virus (MOI = 40) were transfected into MCF-7 cells using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's protocol.

BC cell lines (MCF-7, MCF-7/ADR, BT-549, BT-549/ADR, MDA-MB-468 and SKBR-3) were purchased from Procell Company (Wuhan, China). MCF-7 cells were cultured in DMEM (Gibco, USA) and BT-549 cells were RPMI 1640 (HyClone, USA). MCF-7/ADR cells were grown in DMEM containing 10% FBS and1 µg/mL ADR. BT-549/ADR cells were grown in RPMI 1640 containing 10% FBS and1 µg/mL ADR. MDA-MB-468 cells were cultured in DMEM/F12 medium with 10% FBS. SKBR-3cells were plated in DMEM supplemented with 10% FBS. All media were supplemented with 10% fetal bovine serum (BI, Israel) and 1% penicillin/streptomycin (HyClone, USA). The cells were grown in a 37°C humidified incubator with 5% CO₂. EZR siRNAs (si-EZR), scrambled negative control (NC) siRNAs (si-NC), empty pcDNA3.0 vector and EZR-pcDNA3.0 vector were synthesized by Gene Pharma (Shanghai, China), as follows: EZR 5′-UUCUCAUAAAUAUUCAGUCCAAGGG-3′, 5′-UUCUGCGTACCUAUCACGUTT-5′. Briefly, cells were plated in six-well plates at a density of 5 × 10⁵ cells/well overnight and then transfected with 50 nm si-EZR or si-NC by using Lipofectamine 3000 reagent (Invitrogen, USA).

The TNBC cell lines (MDA-MB-231, MDA-MB-468, MDA-MB-453, and BT-549), normal mammary gland cell line MCF-10A, ER⁺/HER2⁻ cell line MCF-7¹¹ , and ER⁺/HER2⁺ cell line BT-474¹¹ were commercially obtained from the Procell Life Science&Technology Co., Ltd (China). MDA-MB-231, MDA-MB-468, and MDA-MB-453 were maintained in Leibovitz’s L-15 medium supplemented with 10% FBS at 37 °C. BT-549 were maintained in RPMI-1640 medium supplemented with 10% FBS (Solarbio, China) in 5% CO₂ ambiance at 37 °C.
For circBACH2 overexpression, pLCDH-ciR vector (Geneseed, China) containing the cDNA of circBACH2 was employed (named as circBACH2). Mock vector was used as a negative control (named as vector). For circBACH2 silencing, pLL3.7 vector (FENGHUISHENGWU, China) containing the shRNA against circBACH2 was employed (named as sh-circBACH2). ShRNA-NC was served as a negative control (named as sh-NC). miR-186-5p/miR-548c-3p inhibitor and miR-186-5p/miR-548c-3p mimic were provided by RiboBio (China) commercially. Cell transfection was implemented with the assistance of Lipofectamine 3000 (Thermo Fisher, USA). Forty-eight hours later, the transfection efficiency was confirmed through qRT-PCR.

Plasmids of pcDNA3-SIPA1, pcDNA3-N-SIPA1 encoding N-terminal half part of SIPA1 (1-539aa) and pcDNA3-C-SIPA1 encoding C-terminal half part of SIPA1 (540-1042aa) were constructed as previously described (Ma et al., 2021 (link)). SIPA1 and EPAS1 shRNA nucleotides (Supplementary Table S1) were cloned into pLKO.1-GFP (RRID: Addgene_30323) to construct pLKO.1-GFP-SIPA1 and pLKO.1-GFP-EPAS1, respectively. Lentivirus packaging plasmids, psPAX2 (RRID: Addgene_12260) and pMD2. G (RRID: Addgene_12259), and a reporter plasmid pRL-TK (RRID: Addgene_11313) were purchased from Merck & Co. (Kenilworth, NJ). EPAS1 promoter region (-1618bp to transcription starting site) was cloned into pGL4 (RRID: Addgene_48744) to give pGL4-EPAS1. pGEX-C-SIPA1 was constructed to express GST-tagged C-terminal half part SIPA1 protein.
Human cell lines MDA-MB-231, MCF7, and HEK293T were purchased and authorized from China Center for Type Culture Collection (CCTCC, Wuhan, China). BT549 was purchased from Procell (Wuhan, China) with short tandem repeat authentication. SUM159 were gifted from Dr. Peijing Zhang (Yao et al., 2018 (link)). Cells were cultured according to the previous study (Ma et al., 2021 (link)). The stable knockdown cells were established following the previous study (Zhang et al., 2015 (link)).