EGF treatment promotes recovery of human HSCs following irradiation. (A) Immunofluorescence microscopy of γ-H2AX foci in nonirradiated and 300-cGy–irradiated human BM CD34⁺ cells cultured in complete media (TSF) with and without 100 ng/mL EGF for 1 hour (scale bar, 10 µm). Numbers of foci per cell are quantified (n = 24-27 fields of view, 1-way ANOVA) (left panel). Nonirradiated (Non-IRR) or 300-cGy–irradiated human BM CD34⁺ cells were treated with EGF or saline for 5 minutes and analyzed for p-EGFR (B), p-Akt (C), p-DNA-PKcs (D), and p-Artemis (E) levels (n = 4 per group, 2-way ANOVA). (B) Representative flow cytometric analysis of p-EGFR in BM CD34⁺ cells (left panel). Quantification of the percentage of p-EGFR (right panel). (C) Representative analysis of p-Akt in BM CD34⁺ cells (left panel). Quantification of the percentage of p-Akt (right panel). (D) Representative analysis of p-DNA-PKcs in BM CD34⁺ cells (left panel). Quantification of the percentage of p-DNA PKcs (right panel). (E) Representative analysis of p-Artemis in BM CD34⁺ cells (left panel). Quantification of the percentage of p-Artemis (right panel). (F) XRCC6 gene expression in human BM CD34⁺ cells following 300-cGy irradiation and treatment with EGF or saline for 1 hour. Non-IRR BM CD34⁺ cells cultured for 1 hour were used as control (n = 3, mean ± SEM, 1-way ANOVA). (G) Representative flow cytometric analysis of human CD34⁺CD38⁻ HSPCs in culture at 36 hours following 300-cGy irradiation of BM CD34⁺ cells and treatment with media with or without EGF and with or without NU7441 (left panel). Mean percentages of CD34⁺CD38⁻ cells (n = 5 per group, 1-way ANOVA) (right panel). (H) Numbers of CFCs after 36 hours of culture of BM CD34⁺ cells following 300-cGy irradiation and treatment with or without EGF and with or without NU7441 (n = 6, mean ± SEM, 2-way ANOVA). (I) Representative human CD45⁺ cell engraftment in PB of NSG-S mice at 16 weeks following transplantation of irradiated BM CD34⁺ cells cultured in media + EGF for 36 hours or irradiated BM CD34⁺ cells cultured in media alone (left panel). Time course of human CD45⁺ hematopoietic cell engraftment in PB of NSG-S mice (n = 7 or 8, mean ± SEM, 2-way ANOVA) (right panel). Time course of human CD33⁺/CD13⁺ myeloid cell engraftment (J), human CD19⁺ B-cell engraftment (K), and human CD3⁺ T-cell engraftment (L) in NSG-S mice (n = 7 or 8, mean ± SEM, 2-way ANOVA). *P < .05, **P < .01, ***P < .001, ****P < .0001.