p97 is typically recruited to ubiquitinated proteins by cofactors containing ubiquitin binding domains. Among the large number of p97 cofactors involved in many different cellular processes, we focused on those that were reported to function in the nucleus. siRNAs to UFD1, NPLOC4, UBXN6, FAF2, and PLAA were used to deplete the respective proteins in U2OS PML−/− + YFP-PML cells. In this background, arsenic-induced degradation of PML over a 24-h period was followed by Western blotting (Fig. S3 A). In all cases, knockdown of the target protein was effective, although as expected, knockdown of NPLOC4 also led to the depletion of its heterodimeric partner UFD1 (Fig. S3 B). In cells treated with non-targeting (NT) siRNA, arsenic exposure led to the accumulation of higher molecular weight PML forms which were reduced after 6 h and almost completely depleted after 24 h. While knockdown of UBXN6, FAF2, and PLAA failed to block PML degradation, knockdown of UFD1 and NPLOC4 led to reduced degradation of PML and accumulation of higher molecular weight forms of PML, even after 24 h of arsenic treatment (Fig. S3 A). To confirm these findings and determine the consequences of siRNA-mediated depletion of p97 and associated cofactors on PML body number, size, and PML content, U2OS PML−/− + YFP-PML cells were treated with siRNA for 48 h before exposure to arsenic for a further 24 h. Cells were analyzed by Western blotting at 0, 6, and 24 h after arsenic addition and by fluorescence microscopy after 0 and 24 h. Even prior to arsenic treatment, Western blotting shows that knockdown of p97 leads to the accumulation of PML (Fig. 5 A). Although less pronounced, this is also evident with knockdown of UFD1 and NPLOC4. Unexpectedly, knockdown of UBXN6 leads to a reduction in PML levels prior to arsenic treatment, suggesting it has a positive influence on PML body numbers under normal conditions (Fig. 5 A and Fig. S3 A). In response to arsenic, knockdown of p97, UFD1, and NPLOC4 appears to block PML degradation. In contrast, knockdown of UBXN6 appears to enhance arsenic-induced degradation of PML (Fig. 5 A). Analysis of YFP-PML by fluorescence microscopy indicates that cells treated with NT siRNA show the expected reduction in PML bodies in response to 24 h arsenic treatment. In contrast, cells treated with siRNA to p97, UFD1, and NPLOC4 and exposed to arsenic accumulate PML in large intense nuclear bodies, which is not the case for UBXN6 (Fig. 5 B). To quantify arsenic-induced PML degradation after siRNA treatment, time-lapse microscopy was used to monitor YFP-PML fluorescence over a 10-h period. 10 videos were collected for each siRNA treatment and the quantitative pipeline described above was used to follow PML bodies over time (Videos. 5, 6, 7, 8, 9, and 10). In cells treated with NT siRNA, YFP-PML was depleted in a time-dependent fashion in response to arsenic (Fig. 5 C), reaching >80% depletion after 9.75 h (Fig. 5 D). siRNA to the cofactor UBXN6 had no apparent effect. In cells treated with siRNA to p97, UFD1, or NPLOC4, the rate of YFP-PML depletion was reduced (Fig. 5 C), with final depletion after 9.75 h reaching only 40% for siRNA to p97 and 60% for siRNAs for UFD1 or NPLOC4. In all measured PML body metrics, UBXN6 depletion had no significant effect, showing similar data to non-treated cells for PML body numbers, size, and area after arsenic treatment (Fig. S4, A–C). However, cells exposed to siRNA for UFD1 or NPLOC4 showed blunted responses to arsenic treatment by the same measurements (Fig. S4, A–C). This was also true for siRNA for p97, except in this case PML body area increased upon arsenic treatment. These data suggest that the heterodimer of UFD1 and NPLOC4 is the cofactor that is required for arsenic-induced degradation of PML.