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. 2020 Oct 13;23(11):101664. doi: 10.1016/j.isci.2020.101664

Figure 4.

Figure 4

Condensin II Accumulates at nucSF and Associates with RepoMan in Arsenite-Stressed Cells

(A) Diagram detailing the complementary SILAC-based quantitative AP/MS and BioID strategies used to compare the interactome of RepoMan in arsenite-stressed vs untreated cells. For AP/MS, untreated cells were labeled with heavy (H) media and arsenite-treated cells with light (L) media. Endogenous GFP-tagged RepoMan in the HEK293 knock-in cell line was immunoprecipitated using the GFP-Trap_A affinity resin and associated proteins identified by MS analysis. For BioID, the labeling strategy was flipped so that untreated cells were labeled with light media and arsenite-treated cells with heavy media. BioID2-tagged RepoMan was lentivirally transduced in U2OS cells and biotinylated proteins captured on streptavidin affinity resin and identified by MS analysis. ARS:UT ratios were determined for all proteins identified (L:H for AP/MS, (H)L for BioID).

(B) The table highlights overlapping and novel hits that showed increased association with RepoMan in single replicates of the internally controlled complementary screens (full data sets provided as Table S3). The number of peptides detected for each protein is listed, as well as its ratio ARS:UT (with the median ratio for the experiment listed for comparison). Note that in the AP/MS experiment NCAPD3 and NCAPG2 peptides were only detected in the L (+ARS) form. Asterisk (∗) indicates that MaxQuant did not calculate a SILAC ratio for the protein, so enrichment was estimated by comparing the summed L vs. H peptide intensities.

(C) AP/Western blot validation of the increased association of endogenous NCAPD3 with endogenous GFP-tagged RepoMan in arsenite-stressed HEK293 knock-in cells.

(D) BioID/Western blot validation of increased biotinylation of endogenous NCAPD3 by transduced BioID2-RepoMan in U2OS cells. Both blots were probed with anti-NCAPD3.

(E) Live imaging of U2OS cells stably expressing GFP-NCAPD3, either untreated (left panel) or treated with 0.5 mM arsenite for 30 min (arrows indicate stress-induced nuclear foci).

(F) Live imaging of U2OS cells stably expressing GFP-NCAPD3 (green) and transiently expressing mCherry-RepoMan (red), treated with 0.5 mM arsenite for 30 min to induce nucSF (red arrow) at which NCAPD3 (green arrow) accumulates. The inset panels show the individual NCAPD3 (top panel) and RepoMan (bottom panel) signals. Scale bars are 5 μm.