Figure 2

Ectopic expression of FAAP100^WT or FAAP100^T542P in FAAP100-deficient cells.

(A) Metaphase micrographs of FAAP100^T542P-mutant 1176 cells after exposure of the cultures to MMC (100 ng/mL, 48 hours). Parental 1176 cells and mock- or mutation-transduced (+vector^pLVX or +FAAP100^T542P) 1176 cells show distinctly increased chromosome breakage, mostly of the chromatid type, whereas WT transduced 1176+FAAP100^WT cells are rescued. Radials are marked with red arrows. (B) Box plots reflect the proportion of cells with the indicated number of chromosome breaks per metaphase; single value (♦), median (─), mean (□), IQR (─), minimum (×), and maximum (×) for the number of breaks from 3 independent experiments; blue symbols are from untreated cultures, and red are from cultures exposed to MMC (100 ng/mL, 48 hours). Light gray shading indicates high rates of 8 or more breaks per metaphase, and red arrows highlight pivotal rates of 10 or higher. Cell lines are the same as in A. Fifty metaphases were analyzed per experiment. (C) Cell-cycle analysis by flow cytometry. Exemplary individual measurements. Black histograms are from untreated cultures, and superimposed gray or gray/red are from cultures exposed to MMC (10 ng/mL, 48 hours). The percentage of cells in the G₂ phase is indicated. Red coloring and arrows denote an increased G₂ compartment size. Variability and significance of G₂ phase arrest in repeated measurements are shown in Supplemental Figure 4A. Cell lines are the same as in A. (D and E) Dose-response (survival) curves of parental and mock FAAP100^WT or FAAP100^T542P transduced 1176 cells from cultures exposed to different concentrations of MMC (D) or CDDP (E) for 8 days. Data indicate the mean ± SD of 3 independent experiments. Cell lines are the same as in A and are identical in D and E. FA-B and non-FA are FA and normal control, respectively. LC₅₀, 50% lethal concentration. Note that the transduction of FAAP100^WT complements the repair defect in all assays, whereas FAAP100^T542P does not.