Gemcitabine

Chemotherapeutic drugs, VP-16, DXR, PTX, CDDP, carboplatin, gemcitabine, 5-FU, cyclophosphamide, capecitabine, and vincristine were purchased from MedChemExpress. Topo IIα (catalog 12286), Smurf2 (catalog 12024), cPARP (catalog 5625), and Bim (catalog 2933) antibodies were purchased from Cell Signaling Technology Inc. Mcl-1 (sc-12756) and Topo IIβ (sc-55330) antibodies were purchased from Santa Cruz Biotechnology. Anti–phospho-histone H2AX (Ser139; γ-H2AX) antibody was purchased from MilliporeSigma (catalog 05–636). DAPI (catalog 62248) and secondary antibody Alexa Fluor 488 donkey anti-mouse (A32766) were purchased from Thermo Fisher Scientific. Other reagents and antibodies were the same as described previously (10 (link), 16 (link)).

The polyclonal anti-CNT1 antibody was previously generated and characterized in our laboratory [20 (link)]. The CNT1-G7 antibody was purchased from Santa Cruz Biotechnologies (Dallas, TX, USA); the anti-actin antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA); Alexa-488 and Alexa-594 were from Invitrogen (Carlsbad, CA, USA); and anti-mouse and anti-rabbit secondary antibodies were purchased from Bio-Rad (Hercules, CA, USA). Gemcitabine was obtained from MedChem Express (Sollentuna, Sweden), and 5-DFUR was purchased from Sigma-Aldrich.

Gemcitabine and paclitaxel were purchased from MedChemExpress LLC. Protease inhibitor cocktail and phosphatase inhibitor cocktail were purchased from APExBIO Technology LLC. Primary antibodies against p-mTOR, mTOR, p-AKT, AKT, p-PI3K, PI3K, MDR1, cleaved caspase-3, Beclin-1, LC3A/B and GAPDH were purchased from Cell Signaling Technology, Inc.

The cholangiocarcinoma cell line QBC939 and HUCCT1 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The human embryonic kidney 293T (HEK293T) cells were purchased from the American Type Culture Collection. QBC939 and HUCCT1 were all MTHFD1 WT genotype (MTHFD1^+/+). QBC939, HUCCT1, and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco). All cell lines were supplemented with 10% fetal bovine serum (Gibco), penicillin (100 mg/ml) and streptomycin (100 mg/ml) and were incubated in a humidified chamber with 5% CO₂ at 37 °C. Gemcitabine, Methotraxate, and puromycin were purchased from MedChemExpress (MedChemExpress, Monmouth Junction, NJ).

Bladder tumor cell lines, UMUC3 and T24, and urothelial cell line, SV-HUC-1, were acquired from the cell bank of the Chinese Academy of Sciences and cultured in high glucose DMEM or PRMI 1640 medium with 10% FBS (Gibco, Thermo Fisher Scientific, USA). All reagents [EPZ015666 (22 nM), GSK3326595 (6.2 nM), gemcitabine (7.5 ng/mL), cisplatin (100 mg/mL)] were purchased from MedChemExpress, and various concentrations FKA were used to treat UMUC3 and T24 cells.

A human normal intrahepatic bile duct cell line (HiBEC cells), two human CCA cell lines (HUCCT1 and RBE cells), the human umbilical vein endothelial cell line (HUVECs) and the 293T cell line were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HiBECs, HUVECs and 293T cells were cultured in DMEM supplemented with 1% penicillin/streptomycin and 10% FBS (medium and supplement from Gibco®, Gaithersburg, MD). The HUCCT1 and RBE cell lines were cultured in RPMI-1640 medium supplemented with 1% penicillin/streptomycin and 10% FBS. The NFs and CAFs isolated from human tissues were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented 1% penicillin/streptomycin and 15% FBS (medium and supplement from Gibco®, Gaithersburg, MD). Transwell chambers were employed in this study to coculture cells (Figure S2A). Gemcitabine was purchased from MedChemExpress (Shanghai, China). The IC50 values of Gemcitabine in the HUCCT1 and RBE cells were detected by CCK-8 assay (Gemcitabine IC50 values: 0.0236 µmol/L in HUCCT1 and 2.51 µmol/L in RBE cells, Figure S2B). Cytokines, including IL-1beta, IL-6, IL-8, VEGF-alpha, CXCL12, and TGF-beta1, were purchased from Beyotime Biotechnology (Shanghai, China).