Optimizing Inhibitor Concentrations in Cell Studies

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)).