Figure 7. FBXO11 promotes Set8-dependent migration of epithelial cancer cells through the targeted degradation of Cdt2.

(A) Representative images of scratch or wound healing assay showing the migration of A549 cells transfected with si-GL2 or si-FBXO11. Cells migrate more efficiently in the presence of FBXO11 as demonstrated 24 hrs following the scratch.

(B) Quantitation of the migration distance traveled (in micrometers) in si-GL2 or si-FBXO11-trasnfected cells without or with TGF-beta for 48 hrs. Error bars represent the average +/− SD from 12 independent scratches (three fields per scratch).

(C) Similar to (B) except that the analysis was performed on A549 cells growing in 2% or 10% serum concentrations. Error bars represent the average +/− SD from 12 independent scratches (three fields per scratch).

(D) Similar to (B, C), but in control A549 cells (pMSCV) or in cells stably expressing wt-Cdt2 or Cdt2 proteins resistant to degradation by FBXO11 (Cdt2^D457A and Cdt2^S462A). See also Figure S5. Error bars represent the average +/− SD from 12 independent scratches (three fields per scratch).

(E, F) Set8 is required for the efficient migration of epithelial cells. (E) Immunoblot of lysates from A549 cell lines from (D) infected with a second retrovirus expressing either wt-Set8 or catalytically inactive Set8 (Set8^R265G/D338A) to show the expression of the indicated Set8 proteins. Anti-flag immunoblot shows the expression of ectopic Set8 proteins. Asterisk represents a cross-reactive band in the anti-Set8 immunoblot. Loading control: tubulin. (F) Similar to (D) except the analysis was performed in the indicated A549 cell lines as described and shown in (E). Migration distance (in micrometers) was determined 48 hrs following the initial scratches. Error bars represent the average +/− SD from 12 independent scratches (three fields per scratch).