Figure 3

FANCD2 monoubiquitylation capacity of FAAP100-inactivated human and avian cell lines.

(A) FAAP100^T542P mutant 1176 cells (parental) lacked FANCD2 monoubiquitylation (◄D2) on immunoblots. FANCD2 monoubiquitylation was restored only in 1176+FAAP100^WT transduced cells (◄D2-Ub), but not in 1176+FAAP100^T542P or mock transduced cells. FA-L was a monoubiquitylation-deficient FA control. (B) FAAP100^T542-mutant 1176 cells showed reduced levels of FAAP100 protein (see ratio to vinculin in parental and +[empty] vector^pLVX lanes) and lacked FANCD2 monoubiquitylation (◄D2) on immunoblots. 1176+FAAP100^WT- and 1176+FAAP100^T542P-transduced cells both overexpressed FAAP100, but FANCD2 monoubiquitylation was restored only in 1176+FAAP100^WT cells (◄D2-Ub). FA-D2 was a control for absent FANCD2, and non-FA was a monoubiquitylation-proficient normal control. (C) FANCD2 monoubiquitylation was functional in HEK293T cells (not shown) and HEK293T cells mock transfected with the Cas9-containing vector PX459 (◄D2-Ub), but not in the FAAP100-inactivated HEK293T CRISPR/Cas9 clone Cr10 (parental) (◄D2), where it was rescued by transduction with FAAP100^WT, resulting in overexpression of FAAP100 (◄D2-Ub), as shown by FANCD2 and FAAP100 immunoblots. (D) FANCD2 monoubiquitylation was functional in parental HAP1 cells (◄D2-Ub), but not in FAAP100-inactivated HAP1 FAAP100^R149Vfs*2 cells (◄D2), where it was restored by transduction with FAAP100^WT, resulting in overexpression of FAAP100 (◄D2-Ub), as shown by FANCD2 and FAAP100 immunoblots. (E) FANCD2 monoubiquitylation was functional in parental DT40 cells (◄D2-Ub), but absent in ΔFAAP100-DT40 cells (◄D2). In the latter, it was rescued by the expression of EGFP-FAAP100^WT (◄D2-Ub), but not by EGFP-FAAP100^T547P (◄D2), which contained the chicken equivalent T547P of human T542P, as shown by FANCD2 and EGFP immunoblots. Note that the strong overexpression of the FAAP100 protein by lentiviral transduction in B–D, where low- and high-exposure blots are shown, was in marked contrast to the transfection in E. Vinculin or RAD50 or tubulin was used as a loading control on all blots. The FAAP100/vinculin ratios were taken from the high-exposure FAAP100 blot. The FANCD2-Ub/FANCD2 ratios semiquantitatively estimated monoubiquitylation.