Regeneration and DNA damage response in macroH2A DKO intestine.

<p>(A) Left: representative images of Ki-67 immmunohistochemistry within macroH2A WT and DKO proximal jejunum 3 days after exposure of mice to 12 Gy whole body γ-irradiation. 10x objective. Right: quantitation of Ki67⁺ nascent crypt foci per mm. N = 3 mice per condition, mean ± SD. (B) Top: post-IR lineage tracing scheme: macroH2A WT or DKO <i>Hopx-CreERT2</i>::<i>Rosa26-LSL-tdTomato</i> mice were injected with 2mg tamoxifen 48h and 24h prior to treatment with 12 Gy whole-body gamma irradiation, and 72h later sacrificed for analysis. Bottom: representative immunofluorescence of tdTomato lineage tracing (red) counterstained with DAPI (blue) within macroH2A WT and DKO crypts 72 hours after γ-irradiation. 30x objective (C) Left: quantitation of tdTomato tracing events per 500μm, N = 3 mice per condition, mean ± SD. Right: quantitation of tdTomato tracing events per 500μm normalized to percentage of <i>Hopx-tdTomato</i>^<i>+</i> ISCs during homeostasis (values in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185196#pone.0185196.g004" target="_blank">Fig 4A</a>), N = 3 mice per condition, mean ± SD. (D) Experimental scheme highlighting the timing of DNA damage and apoptosis analysis (24h post IR) and regeneration and lineage tracing analysis (72h post IR) (E) Left: flow cytometry plots of SSC-A vs. cleaved caspase-3 content within total crypt epithelium or <i>Hopx-tdTomato</i>^<i>+</i> subpopulations of macroH2A WT and DKO proximal jejunal crypt cells 24 hours after γ-irradiation. Right: quantitation of total crypt epithelium CC3 positivity and <i>Hopx-tdTomato</i>⁺/CC3 double positivity as defined by boxed subpopulation on left. N = 3 mice per condition, mean ± SD. *p<0.05, ***p<0.0005, ns = not significant, Student’s <i>t</i>-test. Scale bars = 100μm.