a Complex formation of PDE3A with predicted SLFN12 catalytic mutants was assessed by ectopically expressing V5-tagged wild-type or mutant SLFN12 constructs in HeLa cells, treating with DNMDP for 8 h, immunoprecipitating endogenous PDE3A, and detecting coprecipitated SLFN12 by V5 immunoblot. b DNMDP sensitivity of SLFN12 cDNAs with mutations of predicted RNase catalytic residues in HeLa-res cells lacking endogenous SLFN12 expression. Data are plotted as mean values with error bars indicating +/−standard deviation of four replicates. c Direct measurement of SLFN12 RNase activity. In all, 2 µM of wild-type (WT) or the active site mutant (E200A, E205A) SLFN12 were incubated with 2 µg of human rRNA at 37 °C for 40 min. Wild-type SLFN12 was denatured (WT, Den) by heating at 65 °C for 20 min. Cleaved rRNAs were analyzed on a formaldehyde agarose gel. d Induction of SLFN12 RNase activity by PDE3A-SLFN12 complex formation. 0.25 µM PDE3A and SLFN12 proteins were incubated with DMSO, 12.5 µM DNMDP, 12.5 µM trequinsin, or 12.5 µM estradiol at room temperature for 30 min prior to a 1:10 dilution of complex and incubation with rRNA. Digested rRNAs were analyzed on a denaturing agarose gel. The relative amount of intact 28S rRNA was quantified using the ImageJ software, shown at the bottom of the figure. To detect the complex formation between PDE3A and SLFN12, preincubated recombinant proteins were crosslinked using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Reaction was stopped by adding SDS loading buffer. Proteins were separated on an SDS-PAGE gel and detected by silver staining. e RNase activity of the isolated SLFN12 NTD. Experiment with SLFN12 amino acids 1–347 performed as in d.