Heterozygous loss of Ebf1 results in increased levels of phosphorylated H2AX in B-cell progenitors. (A) Representative western blot out of 3 detecting γH2AX and glyceraldehyde-3-phosphate dehydrogenase from in vitro cultured Wt or Ebf1^+/− pro-B cells. (Lower) Quantification of the signal intensity normalized to the Wt control and represents 3 experiments. The data shown in band intensity for mean ± standard deviation (SD) and statistical analysis was performed using an unpaired Student t test: ***P < .001 (B) Graphs over the relative MFI obtained by flow cytmetric analysis to detect γH2AX and indirect flow analysis of phosphorylated Chk1 (pChk1) and phosphorylated Chk2 (pCHK2) in in vitro-cultured Wt or Ebf1^+/− pro-B cells. The data are normalized toward the Wt control and expressed in fold MFI that represents 3 independent experiments. The statistical analysis was done using an unpaired t test. Mean ± SD; **P < .01 and *P < .05 (C) Immunofluorescence analysis of phosphorylated and nonphosphorylated H2AX (total H2AX) in in vitro-cultured Wt or Ebf1^+/− pro-B cells detected by using rabbit γH2AX monoclonal antibody or rabbit monoclonal total H2AX antibody followed by a specific anti-Alexa Fluor 594 secondary antibody. The nuclei were subsequently stained with DAPI, and the images were captured using an LM Zeiss upright confocal microscope. The quantification panel next to the image displaying the signal intensity was collected from 3 experiments. The statistical analysis was performed using an unpaired t test, and results are plotted as change in fold integrated density compared with Wt; **P < .01 and ***P < .001. (D) Diagram with relative MFI obtained by flow cytometric analysis to detect γH2AX, in ex vivo-isolated Wt or Ebf1^+/− pro-B cells. The data are normalized toward the Wt control and represent 3 experiments, and an unpaired t test was performed. Mean ± SD; **P < .01 (E) Diagrams displaying quantitative RT-PCR data from in vitro-cultured Wt or Ebf1^+/− pro-B cells. The data are normalized to the expression of HPRT in triplicate PCR reactions. An unpaired Student t test was performed for statistical analysis. ***P < .001. Data represent 3 independent experiments as mean ± SD. (F) Frequency of AnnexinV⁺ cells as estimated by flow cytometric analysis from in vitro-cultured Wt or Ebf1^+/− pro-B cells. The cells were gated on a lymphoid gate for live cells. The data represent 5 experiments. The error bar in the panels indicate mean ± SD, and statistical analysis was performed using the Student t test. ***P < .001. (G) Cell cycle status of Wt or Ebf1^+/− pro-B cells using Ki67 and DAPI staining. G₀ cells were scored as Ki67⁻DAPI^low, G₁ Ki67⁺ DAPI^low, and SG₂M as Ki67⁺DAPI^high. Mean ± SD and statistical analysis was performed using the Student t test. *P < .05.