Perturbation in early T-cell differentiation caused by loss of PHF6. (A) Western blot of whole-cell lysates from Phf6^lox/Y;Tie2-cre^Tg/+ or control (Phf6^+/Y) thymi probed with anti-PHF6 antibody, followed by an anti–α-tubulin antibody. Each lane represents thymocytes from 1 animal; a total of 8 animals is shown. (B) Thymocytes per 8-week-old thymus. (C) Gating strategy of early T-cell development. ETP (early thymic progenitor) cells are cKIT^hi cells within the CD44⁺CD25^neg quadrant and are overlaid in red. DN2, DN3, and DN4 populations are defined by CD44 and CD25 expression, as indicated. Mean percentages ± SEM of each population as a proportion of thymocytes are displayed within the plots. There was a significant decrease in the percentage of DN2 and DN3 cells (P = .006 and P = .0009, respectively). (D) Quantification of each T-cell population indicated per thymus. (E) Gating strategy for late T-cell development showing mean percentages ± SEM for each population in the plot as a proportion of thymocytes. Lin^neg refers to lack of CD19, B220, MAC1, GR1, and TER119 expression. (F) Quantification of the numbers of thymic double-positive (DP), CD4⁺, or CD8⁺ cells. In panels B-F, n = 10 controls (Phf6^+/Y;Tie2-cre^Tg/+) and n = 9 Phf6^lox/Y;Tie2-cre^Tg/+ mice for all other populations, with the exception of the ETP population (n = 4 per genotype). The color corresponding to genotype is shown in (B). Data are from 8-week-old mice and were analyzed using the 2-tailed Student t test. Bar graphs are presented as individual data points (each circle represents 1 animal), with mean ± SEM.