Figure 3. E9 Induces Cytotoxicity by Covalently Targeting Cys1039 of CDK12.

(A) Viability curves for E9^S and E9^R NB cells treated with E9 for 72 hr. Error bars represent means ± SD. (B) WB of the indicated proteins in E9^S versus E9^R cells treated with DMSO or E9 at concentrations to which the cells had become adapted (Kelly, 500 nM; SK-N-BE2, 300 nM) for 6 hr. (C) WB of unlabeled CDK12 and CDK7 in E9^S and E9^R cells treated with E9 followed by THZ1 for 6 hr at the indicated doses. Cell lysates were incubated with bio-THZ1 (1 μM) to label unengaged CDKs. (D) CDK12 sequence surrounding the THZ1-interacting Cys1039 in THZ1S and THZ1^R cells showing the single point mutation that results in the substitution of Cys1039 for Phe in Kelly E9^R cells (red box). (E) WB of unlabeled CDK12 and CDK7 in WT and C1039S HAP1 cells treated with E9-R (800 nM) or increasing concentrations of E9 as indicated for 6 hr. Cell lysates were incubated with bio-THZ1 (1 μM) to label unengaged CDKs. See also Figure S6.