F 12k medium

HEK293T, HEK293, UMUC3 and U2OS cells were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM (Gibco, 11,995,065). HeLa, T24, 5637, 786-O, H1299, and KYSE150 cells were obtained from ATCC and cultured in RPMI-1640 medium (Gibco, 11,875,093). SV-HUC-1 cells were obtained from ATCC and cultured in F12K medium (Macgene, CM10025). RT4 cells were obtained from ATCC and cultured in McCoy’s 5 A medium (Sigma-Aldrich, M4892). The medium was supplemented with 10% fetal bovine serum (Gibco, 10,099,141 C). The cells were maintained at 37 ℃ in an incubator with 5% CO₂.
MG132 (HY-13,259), cycloheximide (HY-12,320), hydroxyurea (HU, HY-B0313), gemcitabine (HY-17,026), methotrexate (HY-14,519), chloroquine (HY-17,589 A), CVT-313 (HY-15,339), VE-821 (HY-14,731) and GDC-0575 (HY-112,167) were purchased from MedChemExpress. Thymidine (T1895) and nocodazole (M1404) were purchased from Sigma–Aldrich. N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN, B0938) was purchased from TCI.

The human normal uroepithelial cell line SV-HUC and bladder cancer cell lines T24, 5637 and J82 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). T24 and 5637 cells were cultured in RPMI-1640 medium (Gibco, Shanghai, China), while J82 and SV-HUC cells were cultured in minimum essential medium and F-12K medium (Macgene, Beijing, China), respectively. All media were supplemented with 10% fetal bovine serum (FBS; Sagecreation, Beijing, China) and 1% penicillin and streptomycin (Solarbio, Beijing, China). Cells were grown at 37 in an atmosphere of 5% CO₂, and were tested without mycoplasma.
RNF144A-AS1 siRNA and negative control siRNA oligonucleotides were designed and synthesized by GenePharma (Shanghai, China); the sequences are listed in Supplementary Table 5. The siRNA transfections were performed with 100 nM pooled siRNA and Lipofectamine 2000 (Life Technologies) in accordance with the manufacturer’s instructions.

The paclitaxel was purchased from Wuxi Taxus Pharmaceutical Co., Ltd. (Jiangsu, China). Succinic anhydride (SA), 4-dimethyl aminopyridine (DMAP), 1-hydroxy-benzotriazole (HoBt), and 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Macklin (Shanghai, China). N,N-Diisopropylethylamine (DIPEA) was purchased from Aladdin (Shanghai, China). N-(2-Aminoethyl) maleimide trifluoroacetate salt (MAL) was purchased from J&K Chemical Ltd. (Beijing, China). TAR peptide (CRKKRRQRRRATWLPPR) was synthesized from China Peptide Co., Ltd. (Shanghai, China). Esterase and coumarin-6 were purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China) Rabbit anti-NRP-1 antibody was purchased from Abcam (Cambridge, UK). Rabbit anti-β-actin antibody and secondary antibody (anti-rabbit) were purchased from ProteinTech (Beijing, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from BioFroxx (Einhausen, Germany). Cy5.5 maleimide (non-sulfonated, Cy5.5-MAL) was obtained from APExBIO (Houston, TX, USA). Tubulin-tracker green and DAPI were obtained from Beyotime (Shanghai, China). Regular agarose G-10 was purchased from Biowest (Spain). F-12K medium, heparin and endothelial cell growth supplement (ECGS) were obtained from MacGene (Beijing, China). 1640 medium was obtained from Meilunbio (Dalian, China).