Hy 17559

A human normal hepatocyte line (THLE-2) was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. (Ningbo, China). THLE-2 cells were cultured in BEBM medium (CC3170, Lonza/Clonetics Corporation, Walkersville, MD, USA) supplemented with 5 ng/mL EGF (SRP3027, Sigma‒Aldrich), 70 ng/mL phosphorylethanolamine (HY-N5034, MedChemExpress, Monmouth Junction, NJ, USA), 10% foetal bovine serum (FBS, 10099141, Gibco Laboratories, Gaithersburg, MD, USA) and 1% penicillin–streptomycin solution (C100C5, NCM Biotech, Suzhou, China) in a 5% CO₂ incubator at 37 °C. Human HB cell lines (HepG2 and HuH6) were obtained from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). HepG2 and HuH6 cells were separately cultured in MEM (SH30265.01, HyClone, Logan, UT, USA) or DMEM (C11995500BT, Gibco Laboratories) supplemented with 10% FBS and 1% penicillin–streptomycin solution in a 5% CO₂ incubator at 37 °C. For RNA decay assays, cells were treated with 5 μg/ml actinomycin D (HY-17559, MedChemExpress) for 0, 30, 60, and 90 min. For protein stability assays, cells were treated with 100 μg/ml cycloheximide (HY-12320, MedChemExpress) for 0, 2, 4, and 8 h. For some assays, cells were treated with culture medium (CM) or succinate (S9512, Sigma‒Aldrich) for the desired time.

NSCLC cells were seeded in six-well plates overnight and then treated with actinomycin D (5 μg/ml, HY-17559, MedChemExpress) at 0, 2, 4, and 8 h. Total RNA was then isolated by TRIzol and analyzed by qPCR. The RNA expression for each group at the indicated time was calculated and normalized by β-actin.

293T cells were equally seeded in 5 wells in 24-well plates (5 × 10⁴ cells per well). Then 24 h later, the cells were treated with actinomycin D (2 μg/ml, HY-17559, MedChem Express, Monmouth Junction, NJ, USA) for 0 h, 4 h, 8 h, 12 h, and 24 h, respectively. After that, the cells were harvested, and the relative RNA levels of circ-SMO and linear SMO were analyzed by qRT-PCR and normalized to the values measured in the 0 h group.

CRC cells were seeded in 12-well plates overnight, and then treated with actinomycin D (5 μg/mL, HY-17559, MedChemExpress) at the 0, 3, 6 h. Total RNA was then isolated by TRIzol (15,596,018, Invitrogen) and analyzed by qPCR. The mRNA expression for each group at the indicated time was calculated and normalized by β-Actin. The mRNA half-lives time were estimated according to the linear regression analysis.

First, 293T cells were treated with actinomycin D (2 µg/ml, HY-17559, MedChem Express) for 0, 4, 8, 12 and 24 h. The cells were then harvested at the indicated time points, and the relative RNA levels of circMET and linear MET were analysed by RT-qPCR and normalized to the values obtained for the 0 h group.