Fig 1: Reduced proliferation and evidence of DNA damage in ABCB7-deficient pro-B cells.(A) Intracellular flow cytometry analysis of EdU incorporation and pH2A.X expression in Fr. B (B220+ CD19+ CD43+ BP-1-) and Fr. C (B220+ CD19+ CD43+ BP-1+) cells from wild-type (WT) and Mb1-cre ABCB7 conditional knockout (cKO) mice. Cells were pulsed with EdU for 3 hr in culture. Contour plots are representative of three independent experiments (total of three mice/group). (B) Quantification of the proportion of EdU- cells (left graph) and EdU+ cells (right graph) that were positive for pH2A.X expression. (C) Flow cytometric analysis of the proportion of Fr. B and Fr. C cells (A) that incorporated EdU. Histograms are representative of three independent experiments (total of three mice/group). (D) Quantification of the proportion of cells that incorporated EdU (left plot) and EdU gMFI (right graph) in Fr. B and Fr. C cells from (C). gMFI was normalized to WT Fr. B cells. (E–K) Intracellular flow cytometry analysis of PARP (E), pChk1 (F), total Chk1 (G), p53 (H), pATM (I), and CDK2 (J), and Ki-67 (K) expression in Fr. C cells from WT and Mb1-cre ABCB7 cKO mice. Quantification of the MdFI or percent positive is shown on the right of each plot. Isotype controls are shown in gray. Offset and overlaid histograms are representative of at least three independent experiments (total of 4–10 mice/group). (L) Analysis of cell cycle status using intracellular DAPI staining in Fr. B and Fr. C cells from WT and Mb1-cre ABCB7 cKO mice. Leftmost gate marks cells in G1, middle gate marks cells in S phase, and rightmost gate marks cells in G2/M phases. Values shown above gates were derived from the FlowJo cell cycle analysis modeling tool. Quantification of the proportion of cells in G1, S, and G2/M phases is shown on the right of the plot. Proportions were determined by using the FlowJo cell cycle analysis modeling tool. Offset histograms are representative of six independent experiments (total of 8–10 mice/group). (B, D–L) Error bars represent SEM, and p-values are indicated above the data. Statistics were obtained by using an unpaired Student’s t-test.