Actinomycin d

HBEC and NSCLC cells were transfected with relevant plasmids and cultured for 36 h then treated with 10 mM metformin (D150959, Sigma, Saint Louis, USA) followed by analysis at indicated times. For Actinomycin D (129,935, Millipore, Massachusetts, USA) treatment, A549 cells were transfected with relevant plasmids and cultured for 36 h then treated with 5 μg/mL Actinomycin D followed by analysis at indicated times.

To investigate the influence of de novo protein synthesis, 5 μg/ml of cycloheximide (Calbiochem by Merck KGaA, Darmstadt, Germany) was administrated together with IL-17A and/or polyI:C treatment. To explore the stability of the mRNA, the cells were stimulated with polyI:C overnight (approximately 15 hours) to induce the expression of cytokines. Then, actinomycin D (1 μg/ml; SIGMA, Saint Louis, MO) was added together with IL-17A and/or polyI:C to block further mRNA synthesis, and mRNA was harvested at different time points (0.5, 2, 6 hours) after actinomycin D treatment. BAY11-7082 (InvivoGen, San Diego, CA), an IκB-α phosphorylation inhibitor, was added 1 hour before stimulation with IL-17A, polyI:C, and co-treatment of IL-17A/polyI:C to inhibit IκB-α phosphorylation. Cycloheximide, actinomycin D, and BAY11-7082 were dissolved in dimethyl sulfoxide before use.

Cells were transfected with RNAiMax (Invitrogen, Thermo Fisher Scientific) and 20 nM of the siRNA duplexes 24 h after plating. Specific 19-nt sequences were selected in the coding sequence of vimentin to generate 21-nt sense and 21-nt antisense strands of the type (19 N) TT (N, any nucleotide). The siRNA sequences were purchased from Eurogentec (Liège, Belgium) and are listed in Supplementary Table 4. Cells were harvested for subsequent analyses 48 h after transfection.
For actinomycin D (Sigma-Aldrich) experiments, cells were transfected with Vim Si1 or Ctrl Si1 24 h before the addition of 10 µg/ml actinomycin D in their culture media for different time periods (up to 24 h).

Normal human epithelial GES-1 cells and GC cells BGC-823, AGS, HGC-27, MGC-803, SGC-7901 and MKN-45 were provided by the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI 1640/10% fetal bovine serum (ThermoFisher Scientific, Waltham, MA, USA). For actinomycin D treatment, SGC-7901 cells were treated with actinomycin D from Sigma (St. Louis, MO, USA) at 5 µg/mL for 0, 4, 8, 12 and 24 hours. Subsequently, RNA was extracted for assessing the stability of circPOFUT1 and POFUT1 mRNA. For cisplatin (DDP) treatment, cells were treated with DDP (Sigma) at 2 µg/mL for 12 h [16 (link)].

For actinomycin D treatment, 2 mg/ml actinomycin D or dimethylsulfoxide (Sigma-Aldrich, St. Louis, MO, USA) as a negative control was added into the cell culture medium. For RNase R treatment, about 2 μg of RNAs were incubated for 30 min at 37 °C with or without 3 U/μg RNase R according to the manufacturer’s instruction. Then the RNAs were purified with the RNeasy MinElute Cleanup Kit (Qiagen).

RNA extracted from MDA-MB-231 cells was divided into two parts on average: one for RNase R (Epicentre Technologies, USA) and the other for buffer treatment for control. In the experimental group, 2 μg of total RNA was incubated with RNase R (3 U/ug) for 20 minutes at 37 °C. β-actin was used as an internal control.
For Actinomycin D assay, 1 x 10⁵ cells were seeded into 6-well plates and treated with Actinomycin D (2 mg/L; Sigma, USA). Subsequently, the treated cells were collected at 8, 16 and 24 hours, respectively, for qRT -PCR analysis of circAHNAK18 and AHNAK mRNA.