FAAP20 is required for activation of the FA pathway. (A) Immunoblot showing levels of FANCD2 mono-ubiquitination and FAAP20 in HeLa cells stably expressing shControl or shFAAP20#1 siRNAs. Knockdown of FAAP20 expression reduced levels of mono-ubiquitinated FANCD2 in cells treated with MMC or HU compared with UT cells. (B) Immunofluorescence analysis of FANCD2 nuclear foci. HeLa cells stably expressing shControl showed an induction of FANCD2 foci on MMC and HU treatment; knockdown of FAAP20 by shFAAP20#1 resulted in decreased foci formation. Expression of WT _His6-FLAGFAAP20_shRES/Wt rescues capacity to form foci but mutant _His6-FLAGFAAP20_shRES/D164A does not. The percentage of cells with 5 or more foci was determined by examining at least 150 cells. Data are presented as the average of 3 independent experiments with standard deviations. (C) Bar diagram showing chromosome aberrations analysis data. Human HSC93 lymphoblast cells stably expressing control shRNA (shControl), shFAAP20 or shFAAP20, and _His6-FLAGFAAP20_shRES together were analyzed for diepoxybutane-induced chromosomal aberrations like breaks, gaps, and radials. Compared with shControl cells, shFAAP20 cells showed higher number of aberrations per cell and this phenotype was rescued by expressing wildtype FAAP20 resistant to shRNA. Fifty metaphase spreads were prepared and scored for chromosomal aberrations as described in supplemental Methods. (D) Cell-cycle analysis of PI and RNase-stained HeLa:shControl and HeLa:shFAAP20#1 cells that were left UT or treated with MMC for 24 hours. HeLa:shFAAP20#1 MMC treated cells showed an increased number of cells in G₂ phase compared with HeLa:shControl MMC treated cells. (E) MMC survival curve showing that reduced FAAP20 expression results in increased sensitivity to MMC; control levels of MMC sensitivity are restored by expressing FAAP20_shRES/wt, but not FAAP20_shRES/C170A or FAAP20_shRES/D164A mutants. Data represent percent survival, compared with untreated, MMC-naive cells. Each experiment was performed in triplicate, and mean values are shown with standard deviations, derived by comparing each dose to no MMC (0 value on the x-axis). (F) Immunoblot showing that FANCA is reduced when FAAP20 is knocked down using either of 2 shRNAs. (G) Immunoblot showing FANCA is reduced when FAAP20 is knockdown and the reduced levels can be rescued by expressing either WT (WT) or 1 of 2 FAAP20 mutants (D164A or C170A). (H) Immunoblot showing chromatin association of FANCA, FANCG, and FAAP20. HeLa:shControl cells treated with MMC or HU exhibited increased association of both FANCA and FANCG with chromatin, compared with UT cells (lanes 1-3). In contrast, HeLa cells depleted of FAAP20 showed reduced FANCA-chromatin association (lanes 4-6). Induction of chromatin association of FANCA in FAAP20 depleted cells can be rescued by ectopic expression of WT FAAP20 (lanes 7-9), but not by the FAAP20_D164A mutant (lanes 10-12) suggesting FAAP20-ubiquitin binding activity is required for chromatin association of FANCA and FANCG on DNA damage.