Actinomycin d

U2OS cells (ATCC, Manassas, Virginia, USA) were grown in high glucose Dulbecco’s modified Eagle’s media (DMEM) with pyruvate (Thermo Fisher Scientific, Darmstadt, Germany). RPE-1 hTERT cells (ATCC) were cultured in DMEM:F12 media (Thermo Fisher Scientific). Culture media were supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and penicillin/streptomycin (Thermo Fisher Scientific). Cell lines were tested twice a year for Mycoplasma contamination using the LookOut Detection Kit (Sigma), and all tests were negative.
Cells were treated with DMSO (0.2%; Carl Roth, Karlsruhe, Germany), Nutlin-3a (10 µM; Sigma Aldrich, Darmstadt, Germany), Actinomycin D (5 nM; Cayman Chemicals, Ann Arbor, Michigan, USA), 5-FU (25 μg/ml, Cayman Chemicals), or Doxorubicin (0.2 µg/ml; Cayman Chemicals) for 24 h.

Cells were seeded in triplicate in a 6-well plate with 30 × 10⁴ cells per well and allowed to recover for 24 h before drug treatment. Cells were treated with caspase-8 inhibitor (Z-IETD-FMK, FMK007, R&D Systems), caspase-9 inhibitor (Z-LEHD-FMK, FMK008, R&D Systems) or a pan-caspase inhibitor (Z-VAD-FMK, ALX-260-020-M001, Enzo Life Science) at 40 μM, 40 μM or 50 μM, respectively, for 18 h. Cells were treated with either 10 nM vincristine (Cayman Chemical) and incubated for 72 h, 10 nM actinomycin D (Cayman Chemical) for 48 h, 50 μM etoposide (Acros Organics) for 24 h or 0.5 µg/ml TRAIL (PHC1634,Gibco) for 12 h. For multidrug treatment, combinations of etoposide (50 μM) and vincristine (10 nM), or etoposide (50 μM) and actinomycin D (10 nM) were added to the cells and incubated for 24 h. Cell viability was determined using trypan blue staining, and cell number was counted in three different wells on blinded samples. For DNA methyltransferase inhibition assay, RH30 cells were treated with either DMSO (vehicle) or 5-aza-2′deoxycytidine (Sigma-Aldrich) at 30 µM and 60 µM for 48 h prior to RNA isolation. Culture medium supplemented with fresh drug was changed every 24 h. All assays were performed at least twice to confirm results.

Actinomycin D, BTYNB, DZNep and OTX015 were purchased from Cayman Chemical. The compounds were dissolved in DMSO.

HepG2 cells were planted into six-well plates (3 × 10⁵ cells/well). After 24 h, cells were treated with 5 μg/ml Actinomycin D (Cayman Chemical, Ann Arbor, MI, United States) or DMSO and collected at indicated time points. Total RNA (2 μg) was incubated with 3 U/μg of RNase R (Geneseed, Guangzhou, China) for 20 min at 37°C and 10 min at 70°C. After treatment with Actinomycin D or RNase R, the expression of circFGGY and FGGY mRNA were analyzed by qRT-PCR.

Cells were incubated with 5 μg/ml actinomycin D (11421, Cayman, Ann Arbor, MI, USA) for 0, 3, and 6 h. Total RNA was analyzed by qRT-PCR.