Fig. 4.

FAAP20 deficiency sensitizes cells to ICL damage and leads to genomic instability. (A) Schematic representation of the plasmid substrates used in the eChIP assay. The presence of psoralen-ICL and PCR primer locations are indicated. Relative enrichment of FAAP20 at ICLs was calculated by normalizing comparative concentration from real-time PCR of each sample against that of its input. Error bars represent SD from three independent experiments. CTL, control; XL, cross-linked. (B) Clonogenic survival assay of HCT116 cells, FANCL-deficient cells, and FAAP20-deficient cells following MMC treatment. (C) Whole-cell extracts were prepared from HCT116 cells, FAAP20-deficient cells, or FANCL-deficient cells mock-treated or treated with MMC for 24 h. Western blotting was conducted using indicated antibodies. (D) HCT116 cells, FAAP20-deficient cells, and FANCL-deficient cells were mock-treated or treated with 1 μM MMC for 24 h. Immunostaining was performed using anti-FANCD2 antibody and cells were counterstained with DAPI, as indicated. (Magnification: 100×.) (E) Quantification results were the average of two independent experiments and were presented as mean ± SEM. More than 300 cells were counted in each experiment. (F) HCT116 cells or FAAP20-deficient cells were exposed to a low dose of MMC and then treated with colcemid. A representative micrograph shows radial chromosome formation and chromosome breaks marked by arrows that were observed in FAAP20-deficient cells. (Magnification: 100×.) (G) Quantification of chromosome aberration were the average of two independent experiments using wild-type, FAAP20^−/− cells, and FANCL^−/− cells. The data were presented as mean ± SEM. (H) HCT116 cells or FAAP20-deficient cells were mock-treated or treated with 50 nM MMC. Cell-cycle distributions were analyzed by FACS and presented as percentages of cells in G1, S, or G2/M phases.