Cell counting kit 8 cck 8

Cell proliferation assays were performed using Cell Counting Kit-8 (CCK-8; C6005M; US Everbright; Silicon Valley, CA, USA) and 5-ethynyl-2′-deoxyuridine (EdU; C6015M; US Everbright) kits according to their manufacturer’s instructions. For the cell viability assay, PTC cells were inoculated into 96-well plates at a density of 1,000 cells/well and left to stand with 10 μL of CCK-8 reagent for 2.5 h at 37°C with 5% CO₂, then incubated for 0, 24, 48, 72 and 96 h following transfection. For the EdU assay, 5 × 10⁴ cells were inoculated into a 24-well plate and cultured in complete culture medium. After 24 h, cells were stained and photographed according to the manufacturer’s instructions. For the colony formation assay, the corresponding siRNA was transfected into TPC-1 and KTC-1 cells for 24 h. Then, cells were digested with trypsin and transferred into six-well plates (1000 cells/well) and kept for two weeks for colony formation assays. All experiments were repeated at least three times.

Triterpene glycoside Frondoside A (extracted from Cucumaria frondosa, purity > 98% determined by HPLC) was purchased from Kerafast (Boston, MA, USA). Cell Counting kit-8 (CCK-8) was purchased from US EVERBRIGHT^® Inc. (Suzhou, Jiangsu, China). Hoechst-33258 Staining Kit, Annexin-V-FITC Apoptosis Detection Kit, and Cell Cycle and Apoptosis Analysis Kit were obtained from Beyotime (Zhenjiang, Jiangsu, China). α-MEM medium, fetal bovine serum (FBS), PBS, penicillin (100 Units/mL), streptomycin (100 μg/mL), 0.25% Trypsin (1X), and Nonessential Amino Acid Solution (NAAS, 100X) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). PrimeScriptTMRT reagent Kit with gDNA Eraser (Perfect Real Time), TB Green^® Premix Ex TaqTM II (Tli RNaseH Plus), and RNase Free dH₂O were obtained from TaKaRa Bio (TaKaRa, Dalian, China). Chemotherapetic agent Epirubicin hydrochloride (EPI), a commonly used chemotherapy drugs to treat bladder cancer in the clinic, was purchased from Aladdin (Shanghai, China).

Cell culture reagents including DMEM, trypsin, penicillin/streptomycin, and fetal bovine serum were purchased from Gibco. The silver staining kit (LC6100) was purchased from Thermo Fisher. The pyruvate kinase activity assay kit (MAK072) was purchased from Sigma. Cell counting kit-8 (CCK8) (C6005) was purchased from US Everbright. Lactate colorimetric/fluorometric assay kit (K607-100) was purchased from Biovision. DSS (21555) was purchased from Thermo Scientific. The plasmids of pET28a-PKM2, pET28a-PKM2 C424S, were used as described in Li et al. (17 (link)).