Ham s f 12 k medium

Human ureteral epithelial cells SV-HUC-1, BC cell lines including UM-UC-1, UM-UC-3, RT4, RT112 and T24 were purchased from Procell Life Sciences (Wuhan, China). SV-HUC-1 cells were cultured in Ham’s F-12 K medium (Macgene, China) supplemented with 10% fetal bovine serum (FBS) (Gibco, MA, USA). UM-UC-1, UM-UC-3, RT4, RT112 and T24 cells were cultured in RPMI-1640 medium (Gibco, MA, USA) supplemented with 10% FBS. All cells were cultured in 5% CO₂ at 37 °C.
The si-PFK-1 and PFK-1 overexppressing plasmids and their negative control were synthesized by Gemma Shanghai. UM-UC-1 and RT112 cells were grown at 90% fusion. Transfection was performed using Lipofectamine 3000 regent according to the instructions. The culture medium was changed 6 h after transfection, and PCR was performed to detect transfection efficiency at 24–48 h after transfection.

5637, J82, UM‐UC‐3, T24, SV‐HUC‐1, RT4, NCI‐H1299, A549, MDA‐MB‐468, MDA‐MB‐231, MCF‐7, T‐47D, SW116, SW620 and HEK293T were acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), tested negative for mycoplasma and incubated at 37°C with 5% CO₂. 5637, T24, NCI‐H1299, T‐47D and A549 were cultured in Roswell Park Memorial Institute 1640 medium (Gibco). J82, UM‐UC‐3 and HEK293T were cultured in Dulbecco's modified Eagle's medium (Gibco). SW116, SW620, MDA‐MB‐468 and MDA‐MB‐231 were cultured in Leibovitz's L‐15 medium (Gibco). RT4, SV‐HUC‐1 and MCF‐7 were cultured in McCoy's 5A (Gibco), Ham's F‐12K medium (Macgene, Beijing, China) and Minimum Essential Medium (Macgene), respectively. Medium was added with 10% foetal bovine serum (FBS) (Sagecreation, Beijing, China) and 1% penicillin/streptomycin (Solarbio, Beijing, China).
The transfection of plasmids and siRNAs was performed with Lipofectamine 2000 (Invitrogen, USA) based on the manufacturer's protocols. Lentivirus overexpressing LNPPS or the CRISPR‐dcas9‐KRAB/sgRNAs for silencing LNPPS were transfected into BC cells with 5 mg/ml polybrene for 18 h. Cells were then selected by puromycin (2 μg/ml) or blasticidin S (1 μg/ml). Detailed descriptions of siRNAs and sgRNAs are shown in Table S1.

The biological functions of the most significant lncRNA, AC006160.1, were examined via in vitro experiments. Human cell lines SV-HUC-1 (CL-0222), BIU-87 (CL-0035), HT-1376 (CL-0672), T24 (CL-0227), RT4 (CL-0431), RT-112 (CL-0682), 5637 (CL-0002), and UMUC3 (CL-0463) were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). BIU-87, T24, RT4, RT-112, and 5637 cells were cultured in the Roswell Park Memorial Institute (RPMI)-1640 (Macgene, China) medium supplemented with 10% foetal bovine serum (FBS) (Gibco, MA, USA). HT-1376 and UMUC3 cells were cultured in MEM (Macgene, China) supplemented with 10% FBS (Gibco, MA, USA). SV-HUC-1 cells were cultured in Ham's F-12K medium (Macgene, China) supplemented with 10% FBS (Gibco, MA, USA). All cell lines were cultured at 37°C in a humidified incubator containing 5% CO₂.