Actinomycin d

KYSE140 and EC9706 cells were seeded in six-well plates and treated with 100 ng/mL actinomycin D (Amresco, Solon, OH, USA) to inhibit new RNA synthesis for 0 to 10 h. Then, circHIPK3 and HIPK3 mRNA were detected by RT-qPCR analysis.

The stability of circ_0003074 was analyzed using RNase R (ZhongBeiLinGe Biotechnology, Beijing, China) as well as Actinomycin D (Amresco, Solon, OH, USA). One μg RNA from MG63 and Saos2 cells was pre-incubated with 4 U RNase R in an incubator to digest linear RNA. Additionally, MG63 and Saos2 cells were allowed to grow in the media supplied with 2 μg/mL Actinomycin D for 0, 12, 24 and 36 h, followed by RNA isolation according to the aforementioned method. At last, RNA was subjected to qRT-PCR analysis to confirm circ_0003074 and DHTKD1 relative expression.

The distinct germ cell types were isolated from the testes of ICR mice. GC1-spg cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, USA) and cultured in Dulbecco’s modified Eagle’s medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, NY, USA) and 1% penicillin-streptomycin (Invitrogen, CA, USA) at 37 °C with 5% CO₂. For cell treatment, 100 μM Etoposide (Selleckchem, Houston, TX, USA) was incubated with cells for the periods of time. Actinomycin D (5 μg/mL) was purchased from AMRESCO. Cycloheximide (CHX, 100 μg/mL) was purchased from Sigma-Aldrich (St. Louis, MO, USA). SB203580 (10 μM) was purchased from Selleck Chemicals (Houston, TX, USA).

Gefitinib, tucatinib and MG132 were obtained from MedChemExpress (HY-50895, HY-16069 and HY-13259). Actinomycin D was obtained from Amresco (J608). SP600125 and human EGF were purchased from Sigma-Aldrich (S5567 and E9644). PD153035 was purchased from Selleck (S6546). Cycloheximide was obtained from Beyotime (S1560).
The antibodies against human c-Jun, PARP-1, phosphorylated ERK1/2 (Thr202/Tyr204), phosphorylated mTOR (Ser2448), TSC1, JNK and phosphorylated JNK were from Cell Signaling Technology (9165, 9532, 4370, 5536, 6935, 9252 and 4668). The anti-CNOT3 antibody was purchased from Proteintech (11135-1-AP). The antibodies against phosphorylated c-Jun (S73), ki67 and ubiquitin were from Abcam (ab30620, Ab16667 and ab134953). The antibodies against ERK1/2 were purchased from Abcam (ab54230) and Cell Signaling Technology (4695). The antibody against mTOR was purchased from Santa Cruz Biotechnology (sc-517464). The antibody against Keratin 7 was obtained from ZSGB-BIO (ZM-0071).

The NB cells grown in 12-well plates were subjected to incubation with Actinomycin D (Amresco, Solon, OH, USA) for 0, 4, 8, 12, and 24 h. These cells were subsequently lysed and circRANBP17 consent was quantified by qRT-PCR. Linear RANBP17 acted as a reference.

Northern blots were performed as previously described^{38 (link)}. Briefly, total RNAs were separated on an agarose (1.5%)-formaldehyde (2.2 M) gel. After being transmembraned and crosslinked, the nylon membranes (Solarbio, Beijing, China) were incubated with 5’ digoxin labeled LNA-modified probes targeting both circBNC2 and BNC2 mRNA or specific to circBNC2 or β-actin mRNA separately at 55 °C overnight. The membranes were adequately washed in 2 × SSC buffer (Boster, Wuhan, China) at room temperature and incubated with anti-DIG conjugate followed by visualizing with the chemiluminescence substrate CSPD supplied in DIG Luminescent Detection Kit (Roche) according to the manufacturer’s instructions. The probes were synthesized by Exiqon according to the sequences of the target genes listed in Supplementary Table 2.
To determine the stability of circBNC2, total RNAs (2.5 μg) from HK2 cells was degraded for 20 min at 37 °C with or without 5 U/μg RNase-R (Epicenter Technologies), followed by northern blots. To test the stability of circBNC2, HK2 cells were seeded in six-well plates and treated with 100 ng/ml actinomycin D (Amresco) to inhibit new RNA synthesis for 8, 16 and 24 h. Then, circBNC2 and BNC2 mRNA were detected by qRT-PCR.