AREG is overproduced and induced by persistent DNA damage in Brca2-deficient LepR⁺ stromal cells. (A) Schematic presentation of experimental design. (B) Heatmap of dysregulated cytokines in the knockout mice. Cytokine levels in BM supernatants from the 3 niche-specific Brca2 knockout mouse strains and their WT Ctrs (Brca2^fl/fl) were analyzed using a cytokine assay (n = 2 assays per genotype). Heatmap was ordered based on fold change (⁠$≥$ 1.5). (C) AREG levels in the BM supernatant of LepR-Cre;Brca2^+/+ and LepR-Cre;Brca2^fl/fl mice, as detected by ELISA (n = 6-7). (D) Expression of Areg transcript in BM CD45^–LepR⁺ cells of LepR-Cre;Brca2^+/+ or LepR-Cre;Brca2^fl/fl mice detected by qPCR (n = 6-7). (E) Flow cytometry analysis of BM CD45^–LepR⁺ cell frequencies (top) and absolute numbers (bottom) in LepR-Cre;Brca2^+/+ or LepR-Cre;Brca2^fl/fl mice. The representative flow (top) and quantification of CD45^–LepR⁺ cells frequencies (middle) and absolute numbers (bottom) are shown (n = 6). (F-G) Persistent DNA damage induces AREG expression. BM CD45^–LepR⁺ cells isolated from LepR-Cre;Brca2^+/+ or LepR-Cre;Brca2^fl/fl mice after IR (300 cGy) were subjected to immunofluorescence staining for γ-H2AX foci (F) and qPCR analysis for Areg messenger RNA levels (G) at 0, 2, 8, 16, and 32 hours after IR (n = 6). Results are presented as means ± standard deviation of 3 independent experiments. Two-tailed unpaired t test or Wilcoxon rank sum test was performed to compare LepR-Cre;Brca2^+/+ vs LepR-Cre;Brca2^fl/fl in panels C, D, E, and G; and to compare LepR-Cre;Brca2^+/+ vs LepR-Cre;Brca2^fl/fl at each time point (ie, 0, 2, 8, 16, or 32 hours after IR). For panel F, one-way ANOVA, followed by t tests was performed to compare the indicated time points (2, 8, 16, and 32 vs 0 hours) for each genotype (LepR-Cre;Brca2^+/+ or LepR-Cre;Brca2^fl/fl) separately. ∗P < .05; ∗∗P < .01; ∗∗∗ P < .001. GM-CSF, granulocyte macrophage colony-stimulating factor; HGF, hepatocyte growth factor; IFN, interferon; IL, interleukin; MFG, milk fat globule; TIMP, tissue inhibitor of metalloproteinases; TPO, thrombopoeitin; RAGE, receptor for advanced glycation end products.