VCAM-1 expression on endothelial cells is dependent on ROS and regulates the recruitment of LSKCD34⁻ hematopoietic cells early after transplantation. C57BL/6 recipient mice were treated or not with the antioxidant agent NAC at the time of irradiation. Five minutes after irradiation at 10 Gy, 30 μg of fluorescent anti–PECAM-1 (A), anti–VCAM-1 (B), or anti-IgG isotype antibodies (data not shown) was injected into recipient mice. Twenty-four hours later, mice were not transplanted (A-B top panels) or transplanted (A-B bottom panels) with 1000 LSKCD34⁻ CFSE-labeled cells; and 5 hours after transplantation, videos were performed in the femurs. No signal could be detected with the anti-IgG isotype antibodies (not shown). (A-B top panels) Pictures represent the expression of PECAM-1 (A) or VCAM-1 (B) in selected frames from femoral heads of mice treated or not with NAC. The histogram shows the number of PECAM-1 (A) or VCAM-1 (B) positive frames in the 150 frames that covered femoral heads of mice treated with or without NAC (n = 3 mice). Error bars represent SEM. Statistical analysis was performed with the 1-way analysis of variance test. (A bottom panel) Pictures represent selected frames that show associations of CFSE-labeled LSKCD34⁻ hematopoietic cells and PECAM-1–stained structures in the femoral head. The histogram shows the number of positive CFSE cells in the 150 frames that covered femoral heads of mice treated with or without NAC (n = 3 mice). Error bars represent SEM. Statistical analysis was performed with the 1-way analysis of variance test. (B bottom panel) Pictures represent selected frames that show associations between LSKCD34⁻ hematopoietic cells and VCAM-1–stained structures in the femoral head. The histograms show the number of positive CFSE cells in the 150 frames that covered femoral heads of mice treated with or without NAC (top histogram) and the percentage of CFSE⁺ cells associated with PECAM-1- or VCAM-1–positive frames (bottom histogram; n = 3 mice). Error bars represent SEM. Statistical analysis was performed with the 1-way analysis of variance test. (C) Immunohistochemistry analysis of VCAM-1 expression in irradiated bone marrow of femoral heads of mice treated or not with NAC. Note that fewer sinusoids with intense VCAM-1–labeled cells are present in mice treated with NAC. (D top panel) At 24 hours after a 10-Gy irradiation, FACS analysis of VCAM-1 expression by MECA32⁺CD45⁻ live (Hoechst⁻) cells from femoral heads of mice treated or not with NAC. Note that the fluorescence mean of VCAM-1 expression is similar in the presence or in the absence of NAC but that the number of VCAM-1⁺ cells is different. (D bottom panel) At 24 hours after irradiation at 10 Gy, C57BL/6 recipient mice, treated or not with NAC at the time of irradiation or 24 hours after irradiation, were transplanted with 1000 LSKCD34⁻ cells. One and 9 days after transplantation, FACS analyses were performed and the percentage of MECA32⁺CD45⁻ live (Hoechst⁻) cells expressing VCAM-1 is shown. Statistical analysis was performed with the 1-way analysis of variance test.