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. 2016 Feb 11;5:e10786. doi: 10.7554/eLife.10786

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© 2016, Westernberg et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 6. — (A,B) Mathematically predicted numbers of DN4 (A) and DP cells (B) in Rag2^-/-Itpkb^+/+ (black) or Rag2^-/-Itpkb^-/- (red) mice on the indicated days post α-CD3 antibody injection. >98% of thymocytes in Rag2^-/- mice are DN3 cells (C,D), and progenitor influx within 3 days is negligible. Thus, we set K = 0 in our model for (A,B). (C,D) CD4/CD8 expression on thymocytes (C) and CD44/CD25 expression on DN cells (D) from Itpkb^+/+Rag2^-/- or Itpkb^-/-Rag2^-/- mice before (day 0), or 2 or 3 days post α-CD3 antibody injection. Gates in (D) denote DN1, DN2, DN3, DN3-4 and DN4 cells in clock-wise order, numbers % cells in the DN3 and DN4 gates. Representative of 7 independent experiments. (E) Measured mean ± SEM DN4 cell (upper panel) and ISP (lower panel) numbers in Itpkb^+/+Rag2^-/- (solid line) or Itpkb^-/-Rag2^-/- (hatched) mice before (day 0) or 1, 2 or 3 days after α-CD3 injection. Significance of the indicated comparisons was analyzed as in Figure 2. For DN4 cell numbers, n = 4, 4, 6 or 6 Rag2^-/- and 4, 4, 5 or 5 Itpkb^-/-Rag2^-/- mice, respectively. For ISP numbers, n = 6, 5, 7 or 7 Rag2^-/- and 4, 4, 6 or 5 Itpkb^-/-Rag2^-/- mice, respectively. (F) Mean ± SEM DP cell % (upper panel) or number (lower panel) in Itpkb^+/+Rag2^-/- (solid line) or Itpkb^-/-Rag2^-/- (hatched) mice before (day 0) or 1, 2 or 3 days after α-CD3 antibody injection. Significance of genotype differences per day was analyzed as in Figure 2. n = 6, 5, 7 or 7 Rag2^-/- and 4, 4, 6 or 5 Itpkb^-/-Rag2^-/- mice, respectively. (G) Annexin V (AnnV) staining of DN3, DN3-4, DN4, HSA^hiCD8⁺ ISP or DP cells from uninjected Rag2^-/- (open histograms) or α-CD3 antibody injected Itpkb^+/+Rag2^-/- (black filled histograms) or Itpkb^-/-Rag2^-/-(gray filled histograms) mice two (left) or three (right) days post antibody injection. Representative of 3 independent experiments and 3–4 mice per genotype. Uninjected Rag2^-/- mice contain dying cells in the DN4, CD8-ISP and DP cell gates due to failed β-selection at the DN3 stage. These serve as positive controls for the Annexin V stain. (H) Ki67 expression in DN3, DN3-4, DN4, HSA^hiCD8⁺ ISP or DP cells from α-CD3 antibody injected Itpkb^+/+Rag2^-/- (black filled histograms) or Itpkb^-/-Rag2^-/- (gray filled histograms) mice two (left) or three (right) days post antibody injection. Representative of 2 independent experiments and 3 mice per genotype. Open histograms, day 0 WT isotype control. (I) CD2, CD5, CD71 and CXCR4 surface-levels on, and transgenic Nr4a1/Nur77-GFP expression in DN3 or DN4 thymocytes from uninjected Rag2^-/- (open histograms) or α-CD3 injected Itpkb^+/+Rag2^-/- (black) or Itpkb^-/-Rag2^-/- (gray) mice 2 days post injection. The <1% CD44^-CD25^- negative control cells in uninjected Rag2^-/- mice are non-T cells. Representative of ≥3 independent experiments.

DOI: http://dx.doi.org/10.7554/eLife.10786.009