Figure 1.
Lethal TBI induces mitochondrial damage in BM stromal precursor cells. (A-J) WT mice were lethally irradiated and Sca-1− (A, C, E, G, I) and Sca-1+ (B, D, F, H, J) BM stromal precursors were analyzed at the indicated time points postirradiation. (A-B) The number of Sca-1− and Sca-1+ BM stromal precursor cells in nonirradiated and irradiated mice. Mitochondrial mass (Mitotracker green staining) (C-D), mitochondrial ROS (MitoSox red staining) levels (E-F), mitochondrial transmembrane potential (TMRE staining) (G-H), and glucose uptake levels (I-J) in BM Sca-1− and Sca-1+ BM stromal precursors before and after irradiation. Data are presented as average of 3 to 7 mice per group. (K) Schematic illustration of in vivo irradiation experiment for stromal mitochondria imaging. (L) Representative confocal microscopy images showing mitochondrial (green) network in MSC cultured for 24 hours and 48 hours after in vivo lethal irradiation. The boxed area (a) and the respective high-magnification images show long, tubular mitochondria network in nonirradiated BM MSC. The boxed areas (b-c) and the respective high-magnification images demonstrate global mitochondrial fragmentation with loss of the long, tubular mitochondrial structures and presence of many small rounded mitochondria at 24 hours and 48 hours (red arrows), respectively. (M) Quantification of mean mitochondrial volume per surface in MSC after in vivo irradiation and culture. (N) Frequency of mitochondrial events with size higher or lower than 0.5 μm3 in volume. (O) Schematic illustration of in vitro irradiation experiment for stromal mitochondria imaging. (P) Representative confocal microscopy images showing mitochondrial (green) network in MSC cultured for 96 hours after in vitro irradiation. The boxed area (i) and the respective high-magnification images show long, tubular mitochondria network in nonirradiated BM MSC (similar to nonirradiated L). The boxed areas (ii) and the respective high-magnification images demonstrate again global mitochondrial fragmentation with loss of the long, tubular mitochondrial structures and presence of many small rounded mitochondria at 96 hours after in vitro irradiation of BM MSC (red arrows). (Q) Quantification of mean mitochondrial volume per surface in MSC after in vitro irradiation. (R) Frequency of mitochondrial events with size higher or lower than 0.5 μm3 in volume. Both in vivo and in vitro experiments were performed as 2 independent experiments. Mitochondrial network and volume were analyzed using Imaris surface building algorithm, and the statistical color coding of mitochondrial volume are shown. Scale bar, 2, 3, 5, and 10 µm. (S-U) Schematic illustration of lethally irradiated Dendra2-mito WT mice transplanted with WT BM cells and analyzed at 2 weeks and 1 month posttransplantation (S). Normalized mean fluorescence intensity level of Dendra2 mitochondria in Sca-1− (T) and Sca-1+ (U) BM stromal precursor cells were analyzed before irradiation, and 2 weeks and 1 month posttransplantation. Data are the average of 3 to 6 mice per group. All data are represented as mean ± SEM. Statistical significance was assessed using 1-way ANOVA except in panels Q and R where the 2-tailed Student t test was used. *P < .05, **P < .01, ***P < .001.

Lethal TBI induces mitochondrial damage in BM stromal precursor cells. (A-J) WT mice were lethally irradiated and Sca-1− (A, C, E, G, I) and Sca-1+ (B, D, F, H, J) BM stromal precursors were analyzed at the indicated time points postirradiation. (A-B) The number of Sca-1− and Sca-1+ BM stromal precursor cells in nonirradiated and irradiated mice. Mitochondrial mass (Mitotracker green staining) (C-D), mitochondrial ROS (MitoSox red staining) levels (E-F), mitochondrial transmembrane potential (TMRE staining) (G-H), and glucose uptake levels (I-J) in BM Sca-1− and Sca-1+ BM stromal precursors before and after irradiation. Data are presented as average of 3 to 7 mice per group. (K) Schematic illustration of in vivo irradiation experiment for stromal mitochondria imaging. (L) Representative confocal microscopy images showing mitochondrial (green) network in MSC cultured for 24 hours and 48 hours after in vivo lethal irradiation. The boxed area (a) and the respective high-magnification images show long, tubular mitochondria network in nonirradiated BM MSC. The boxed areas (b-c) and the respective high-magnification images demonstrate global mitochondrial fragmentation with loss of the long, tubular mitochondrial structures and presence of many small rounded mitochondria at 24 hours and 48 hours (red arrows), respectively. (M) Quantification of mean mitochondrial volume per surface in MSC after in vivo irradiation and culture. (N) Frequency of mitochondrial events with size higher or lower than 0.5 μm3 in volume. (O) Schematic illustration of in vitro irradiation experiment for stromal mitochondria imaging. (P) Representative confocal microscopy images showing mitochondrial (green) network in MSC cultured for 96 hours after in vitro irradiation. The boxed area (i) and the respective high-magnification images show long, tubular mitochondria network in nonirradiated BM MSC (similar to nonirradiated L). The boxed areas (ii) and the respective high-magnification images demonstrate again global mitochondrial fragmentation with loss of the long, tubular mitochondrial structures and presence of many small rounded mitochondria at 96 hours after in vitro irradiation of BM MSC (red arrows). (Q) Quantification of mean mitochondrial volume per surface in MSC after in vitro irradiation. (R) Frequency of mitochondrial events with size higher or lower than 0.5 μm3 in volume. Both in vivo and in vitro experiments were performed as 2 independent experiments. Mitochondrial network and volume were analyzed using Imaris surface building algorithm, and the statistical color coding of mitochondrial volume are shown. Scale bar, 2, 3, 5, and 10 µm. (S-U) Schematic illustration of lethally irradiated Dendra2-mito WT mice transplanted with WT BM cells and analyzed at 2 weeks and 1 month posttransplantation (S). Normalized mean fluorescence intensity level of Dendra2 mitochondria in Sca-1− (T) and Sca-1+ (U) BM stromal precursor cells were analyzed before irradiation, and 2 weeks and 1 month posttransplantation. Data are the average of 3 to 6 mice per group. All data are represented as mean ± SEM. Statistical significance was assessed using 1-way ANOVA except in panels Q and R where the 2-tailed Student t test was used. *P < .05, **P < .01, ***P < .001.

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