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Additional Supporting Information may be found in the online version of this article.

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sc-12-0533_sm_SuppData.pdf83KSupplementary Data
sc-12-0533_sm_SupplFigure1.tif474KSupplementary Figure 1. (A): Representative flow cytometry profiles of MS5, ST2 and ST4.5 cells and primary bulk MSCs stained for CD45, CD44 and CD29 expression. MS5 cells stained for CD127 expression were used as an isotype control. (B): Photograph of primary bulk C57BL/6 mouse MSCs in culture captured using 10X magnification. Flow cytometry profiles and bright-field photograph are representative of one experiment.
sc-12-0533_sm_SupplFigure2.tif187KSupplementary Figure 2. (A): Clonogenic survival assay of mouse MSC lines (MS5 and ST2) γ-irradiated at 2-10 Gy and cultured for 7 days under normoxic and hypoxic (5% O2) conditions as previously described. Error bars represent mean ± SD, n=4. (B): Growth curve of MS5 and ST2 cells cultured for 7 days under normoxic and hypoxic (5% O2) conditions. Error bars represent mean ± SD, n=3. (C): Photographs of MS5 and ST2 colonies generated at 7 days following 10 Gy irradiation under normoxic and hypoxic (5% O2) conditions and stained with Coomassie Blue. Photographs are representative of one of four independent experiments.
sc-12-0533_sm_SupplFigure3.tiff2702KSupplementary Figure 3. Mouse MSCs activate a robust DNA Damage Response to IR treatment. (A): Western blot analysis of ATR expression in control MS5, ST2, Bulk MSCs and ST4.5 cells using β-Tubulin expression as a loading control. (B): Graph of the percentage BrdU labeled G1 MS5 and ST4.5 cells (10 Gy); (C): BrdU labeled S phase MS5 and ST4.5 cells (1 Gy) (D): BrdU labeled G1 MS5 and ST4.5 cells (10 Gy) harvested at 0-72 hrs post IR and stained for BrdU incorporation and DNA content using PI. Error bars represent mean ± SD, n=2 (Control) or n=3 (Irradiated). Western blot images are representative of one of three independent experiments.
sc-12-0533_sm_SupplFigure4.tif128KSupplementary Figure 4. Primary bulk mouse MSCs activate IR-induced DNA damage checkpoints. (A): Representative cytograms of bulk MSCs harvested at 0-72 hrs post 10 Gy irradiation and stained for BrdU incorporation and for DNA content using PI. (B): Graph of the percentage BrdU labeled G1 cells of bulk MSCs at 0-72 hrs post 10 Gy irradiation. Error bars represent mean ± SD, n=2 (Control) or n=3 (Irradiated). (C): Western blot analysis of p53 stabilization in bulk MSCs harvested at 0-4 hrs post 1 and 10 Gy irradiation using β- Tubulin expression as a loading control. Flow cytometry and western blot images are representative of one of three independent experiments.
sc-12-0533_sm_SupplFigure5.tif329KSupplementary Figure 5. Mouse MSC lines resolve DNA double strand breaks. (A): Representative immunofluorescent images of MS5, ST2 and ST4.5 cells at 0-24 hrs post 1 Gy irradiation and stained for DNA content (DAPI), γ-H2AX (green) and Rad51 (red) IRIF captured using 60X magnification. (B): Representative immunofluorescent images of γ- H2AX IRIF in MS5, ST2 and ST4.5 cells at 1 hr post 10 Gy irradiation. (C): Western blot analysis of DNA Ligase IV and β-Tubulin expression in nuclear and cytoplasmic extracts of control MS5, ST2 and ST4.5 cells used in end-joining assay (see Fig.5E). All immunofluorescent and Western blot images are representative of one of three independent experiments.
sc-12-0533_sm_SupplFigure6.tif132KSupplementary Figure 6. Primary bulk mouse MSCs display a heterogenous response to IR-induced DNA damage. (A): Representative immunofluorescent images of bulk MSCs 0- 24 hrs post 1 and 10 Gy irradiation stained for DNA content (DAPI), γ-H2AX foci (Green) and Rad51 (Red) IRIF and captured using 60X magnification. (B): Graph of the average number of γ-H2AX foci per cell (50 cells in total per time-point) at 0, 1 and 24 hrs post 1 and 10 Gy irradiation. Error bars represent mean ± SD, n=2. (C): Western blot analysis of γ- H2AX and H2AX expression in primary bulk mouse MSCs at 0-24 hrs post 1 Gy and 10 Gy irradiation using β-Actin expression as a loading control. Immunofluorescent and Western blot images are representative of one of three independent experiments.
sc-12-0533_sm_SupplFigure7.tif812KSupplementary Figure 7. MSCs retain the capacity to differentiate following IR treatment. (A): Oil Red O staining of control and irradiated (10 Gy) MS5 and ST2 cells cultured in adipogenic induction and maintenance media for 17 days as previously described. White arrows indicate spherical-shaped adipocytes containing triglycerides stained as red droplets using Oil Red O. (B): Alizarin Red S staining of calcium deposition by control and irradiated (10 Gy) MS5 and ST2 cells cultured in osteogenic induction medium for 14 days as previously described. Images were captured using 4X and 10X magnification and are representative of one of two independent experiments.
sc-12-0533_sm_SupplFigure8.tif69KSupplementary Figure 8. Overview of the DNA Damage Response in irradiated mouse mesenchymal stromal cells and mouse CD4+ CD8+ thymocytes (Refer to text). Abbreviations: DNA double strand break (DNA DSB).

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