Flavopiridol hydrochloride hydrate

All inhibitors were resuspended in DMSO to recommended effective concentrations (Bensaude, 2011 (link)). Flavopiridol hydrochloride hydrate (F3055, Sigma‐Aldrich) was resuspended to a stock concentration of 12.5 mM and diluted 1:12,500 to an effective concentration of 1 μM. Actinomycin D (A1510, Sigma‐Aldrich) was resuspended to an initial concentration of 1 mg/ml and diluted 1:200 to an effective concentration of 5 μg/ml. Triptolide (T3652, Sigma‐Aldrich) was resuspended to a stock concentration of 10 mM and diluted 1:20,000 to an effective concentration of 500 nM. The effectiveness of all inhibitors was verified on the basis of Pol II phosphorylation changes at the whole nucleus level (Appendix Fig S14). Alpha‐amanitin (A2263, Sigma‐Aldrich) was micro‐injected into the yolk (1 nl per embryo) at a concentration of 0.2 mg/ml (dissolved in water) at the 1‐cell stage (Joseph et al, 2017 (link); Hilbert et al, 2021 (link)). Whole embryo flavopiridol treatment was carried out by adding 10 μM flavopiridol to the embryo media (Vopalensky et al, 2018 (link)).

3T9^MYC-ER fibroblasts were obtained by infecting 3T9 c-Myc flox/flox immortalized fibroblasts (Trumpp et al. 2001 (link)) with a pBabe-Bleo retrovirus encoding the MYC-ER chimera (Sabò et al. 2014 (link)) and were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin/streptomycin. These cells are not listed in any database of commonly misidentified cells and are negative for mycoplasma. Cells were used at subconfluent cell densities for all experiments. Due to infection with the pBabe bleomycin MYC-ER construct, cells are resistant for zeocin. Upon thawing, cells were maintained for 7–10 d in zeocin-containing medium (100 µg/mL) but grown without zeocin for subsequent experiments. MYC-ER activation was achieved by addition of 400 nM of the synthetic 4-hydroxytamoxifen (OHT; Sigma-Aldrich). For RNA degradation validation, cells were treated with 1 µM flavopiridol hydrochloride hydrate (Sigma-Aldrich) for the indicated time points.