March5 (a), Mtch2 (b), or Ube2k (c) were deleted from Bax−/−Bak−/− MEFs by CRISPR-Cas9 gene targeting. DNA sequencing was performed to confirm indel generation at sgRNA target sites (Supplementary Table S2). Confirmed knockout clones were then engineered to express HA-NOXA. The ability of NOXA to trigger MCL1 degradation was abrogated in the knockout cells. MARCH5 is not required for MCL1 degradation in MEFs subjected to DNA damage or protein synthesis inhibition. Bax−/−Bak−/− and Bax−/−Bak−/−March5−/− MEFs were exposed to (d) UV radiation (200 J/m2; then cultured for 4 h), (e) etoposide (50 μM for 16 h; ETOP), or (f) the protein synthesis inhibitor cycloheximide (50 μg/mL for up to 6 h; CHX). Asterisks denote MCL1Matrix, a truncated form of MCL1 that appears to be absent from cells expressing wild-type HA-NOXA for the reasons outlined in the legend to Fig. 1.