<p>MARCHF5 requires its ubiquitin ligase and dimerization activities in PEL.</p>

BCBL-1 iCas9 cells expressing gRNAs targeting <i>MARCHF5</i> or <i>AAVS1</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. To visualize the coMARCHF5 expressed from the lentivirus, cells were treated with 2.5 μM MG132 for 8 hour prior to harvesting for Western blotting.

B. Quantification of Western blots in panel A normalized to total protein stain and g<i>AAVS1.</i> C. Cumulative live cell counts (relative to control gAAVS1) of BCBL-1 iCas9 cells in panel A after the addition of Dox (no MG132). Cells were seeded at 3 x 10⁵ cells/mL and Cas9 expression was induced by Dox treatment (0.5 μg/mL). Every three days, total live cells were counted by trypan blue exclusion assay and split back to 3 x 10⁵ cells/mL to allow the cells to continue proliferating.