Figure 4.
EGFR signaling is necessary for HSC DNA repair and hematopoietic regeneration following TBI. (A) Representative line graphs of p-EGFR levels in BM KSL cells from EGFR-WT (WT) mice and EGFR-DN (DN) mice at 45 minutes following EGF treatment (left panel). Percentage of p-EGFR+ KSL cells in each condition (n = 6 per group, mean ± SEM, 2-way ANOVA) (right panel). (B) Representative flow cytometric analysis of percentages of BM KSL cells and CD150+CD48−CD41− KSL HSCs in WT and DN mice at day +10 following 500-cGy TBI (left panels). Percentages of KSL cells and CD150+CD48−CD41− KSL cells in each group (n = 14 per group, mean ± SEM, Student t test) (right panels). (C) Percentages of total donor GFP+ cells, myeloid cells, B cells, and T cells in the PB of recipient mice at 16 weeks following transplantation of 2.5 × 104 GFP+ KSL cells collected from WT or DN mice at day +10 following 500-cGy TBI, coupled with 2 × 105 BM competitor cells (n = 6 or 7 per group, Student t test). (D) Percentage survival of recipient F1 mice over 30 days following 900-cGy TBI and transplantation with 5 × 105 BM cells collected from EGFR-DN or EGFR-WT mice at 24 hours post-500-cGy TBI (n = 14 mice per group, log-rank test). (E) Representative microscopic image of γ-H2AX foci in nonirradiated and 300-cGy–irradiated BM KSL cells from WT and DN mice cultured with TSF for 1 hour (scale bar, 10 µm) (left panel). Foci numbers per KSL cell (n = 20 fields of view per group, mean ± SEM, 2-way ANOVA) (right panel). (F) Microscopic images of p-Akt in KSL cells from WT and DN mice at 5 minutes following irradiation with 300 cGy and culture in media with or without EGF (scale bar, 10 µm) (left panel). p-Akt MFI in KSL cells from each condition (n = 56-69 cells per group, mean ± SEM, 2-way ANOVA) (right panel). (G) Representative line graphs of p-DNA-PKcs levels in KSL cells from WT and DN mice at 1 hour following 100 cGy (left panel). Percentage of p-DNA-PKcs+ KSL cells in each condition at 1 hour postirradiation (n = 5 or 6 per group, 2-way ANOVA). *P < .05, **P < .01, ***P < .001, ****P < .0001. DAPI, 4′,6-diamidino-2-phenylindole; IgG, immunoglobulin G; IRR, irradiated; Non-IRR, nonirradiated.

EGFR signaling is necessary for HSC DNA repair and hematopoietic regeneration following TBI. (A) Representative line graphs of p-EGFR levels in BM KSL cells from EGFR-WT (WT) mice and EGFR-DN (DN) mice at 45 minutes following EGF treatment (left panel). Percentage of p-EGFR+ KSL cells in each condition (n = 6 per group, mean ± SEM, 2-way ANOVA) (right panel). (B) Representative flow cytometric analysis of percentages of BM KSL cells and CD150+CD48−CD41− KSL HSCs in WT and DN mice at day +10 following 500-cGy TBI (left panels). Percentages of KSL cells and CD150+CD48−CD41− KSL cells in each group (n = 14 per group, mean ± SEM, Student t test) (right panels). (C) Percentages of total donor GFP+ cells, myeloid cells, B cells, and T cells in the PB of recipient mice at 16 weeks following transplantation of 2.5 × 104 GFP+ KSL cells collected from WT or DN mice at day +10 following 500-cGy TBI, coupled with 2 × 105 BM competitor cells (n = 6 or 7 per group, Student t test). (D) Percentage survival of recipient F1 mice over 30 days following 900-cGy TBI and transplantation with 5 × 105 BM cells collected from EGFR-DN or EGFR-WT mice at 24 hours post-500-cGy TBI (n = 14 mice per group, log-rank test). (E) Representative microscopic image of γ-H2AX foci in nonirradiated and 300-cGy–irradiated BM KSL cells from WT and DN mice cultured with TSF for 1 hour (scale bar, 10 µm) (left panel). Foci numbers per KSL cell (n = 20 fields of view per group, mean ± SEM, 2-way ANOVA) (right panel). (F) Microscopic images of p-Akt in KSL cells from WT and DN mice at 5 minutes following irradiation with 300 cGy and culture in media with or without EGF (scale bar, 10 µm) (left panel). p-Akt MFI in KSL cells from each condition (n = 56-69 cells per group, mean ± SEM, 2-way ANOVA) (right panel). (G) Representative line graphs of p-DNA-PKcs levels in KSL cells from WT and DN mice at 1 hour following 100 cGy (left panel). Percentage of p-DNA-PKcs+ KSL cells in each condition at 1 hour postirradiation (n = 5 or 6 per group, 2-way ANOVA). *P < .05, **P < .01, ***P < .001, ****P < .0001. DAPI, 4′,6-diamidino-2-phenylindole; IgG, immunoglobulin G; IRR, irradiated; Non-IRR, nonirradiated.

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