(A) Clonogenic assay of control SPRTN, and SPRTN-KO MEFs. Control SPRTN MEFs were treated with 4-OHT for 24 hr to induce CRE-mediated excision of SPRTN. Cells were then treated with formaldehyde, Etoposide or CPT at the indicated drug concentrations for 24 hr (72 hr for Etoposide) and subsequently allowed to form colonies for 10 days. Cells were plated in triplicate. Error bars represent S.E.M. (B) In vivo complex of Enzyme (ICE) assay. Schematic illustration of the method used to isolate Etoposide-induced covalent Top2 DNA-protein crosslinks from cells. (C) ICE assay of control SPRTN MEFs, SPRTN-KO MEFs, and conditional SPRTN MEFs reconstituted with SPRTN, SPRTN-E112A, SPRTN-Y117C, or SPRTN-∆C. Cells were either untreated or treated for 30 min with 25 uM Etoposide and immediately processed for ICE assay or processed 1 and 2 hr after washing out Etoposide (recovery). 1 ug of DNA was loaded for each time-point per cell line. Membrane was probed with an antibody against Top2 and subsequently re-probed with anti-thymine dimer antibody after 1 min UV treatment to control for DNA loading. Representative Western blot indicating SPRTN levels after reconstitution of SPRTN-KO MEFs.