<p>MARCHF5 protein levels are autoregulated.</p>

BCBL-1 iCas9 cells expressing gRNAs targeting <i>MARCHF5</i> or <i>AAV S1</i> were transduced with lentiviruses constitutively expressing cDNAs for mCherry, codon-optimized MARCHF5 (coMARCHF5) wildtype, H43W ligase mutant, or 4GL dimerization mutant and treated with Doxycycline (Dox) (0.5 μg/mL) for 9 days and harvested for Western blotting.

B. Unedited BCBL-1 iCas9 g<i>AAV S1</i> cells expressing mCherry and coMARCHF5–3XFLAG constructs (no Dox) were left untreated or treated with 2.5 μM MG132 for 8 hours prior to harvesting for Western blotting of endogenous and 3XFLAG-tagged MARCHF5. C. Cumulative live cell counts from the same growth curves of edited BCBL-1 iCas9 g<i>AAV S1</i> cells in Fig 4C were replotted relative to the matched mCherry cells to compare the impact of the cDNAs on cellular viability.