Figure 7
Figure 7. Intracellular ROS in AKT1−/−AKT2−/− LSK cells in vivo and their colony-forming capacity in vitro after pharmacologic increase of ROS. (A-B) ROS content of AKT1−/−AKT2−/− LSK cells. The histogram (A) represents one experiment, and the bar graph (B) represents the mean ± SEM (n = 5) of the MFI of the DCF-DA–treated cells normalized to the HBSS-only–treated cells (no DCF-DA) from each experiment. LSK cells were sorted from bone marrow chimeras, incubated with DCF-DA, and analyzed by flow cytometry. (C-D) ROS content in DN3 thymocytes. Thymocytes from 16-week primary competitive bone marrow chimeras were surface stained with CD4, CD8, CD25, and CD45.2. DN3 staged thymocytes were identified by CD4−CD8−CD25+ phenotype. The histogram (C) represents one experiment, and the bar graph (D) represents the mean ± SEM (n = 3) of the DCF-DA MFI of CD45.2+ cells divided by the DCF-DA MFI of CD45.1+ cells in the same tube. (E) Colony-forming capacity of LSK cells treated with BSO. The graphs represent the total number of colonies found in each culture as a percentage of those generated in the absence of BSO (no BSO = 1) and represent the mean ± SEM (n = 5). Equal numbers of freshly sorted LSK cells were plated in methylcellulose cultures with increasing concentrations of BSO for 10 days before analysis. The number of colonies per plate (mean ± SEM) that used AKT1+/+AKT2+/− cells was 0μM BSO 45 ± 3, 0.0167μM BSO 34 ± 3, 0.020μM BSO 26 ± 2, 0.025μM BSO 29 ± 1, 0.050μM BSO 15 ± 2, and 0.100μM BSO no growth and for AKT1−/−AKT2−/− cells was 0μM BSO 32 ± 2, 0.0167μM BSO 38 ± 1, 0.020μM BSO 32 ± 3, 0.025μM BSO 39 ± 3, 0.050μM BSO 23 ± 2, and 0.100μM BSO 3 ± 1. (F) Effect of BSO treatment on LTC-ICs. LSK cells were grown in LTC-IC cultures for 2 weeks in the presence of 0.025μM BSO. Then, equal numbers of cells were harvested from the LTC-IC cultures and plated in methylcellulose in the absence of BSO for 10 days. The graphs represent the total number of colonies found in each methylcellulose culture as a percentage of those generated in the absence of BSO (no BSO = 1) and represent the mean ± SEM (n = 3).

Intracellular ROS in AKT1−/−AKT2−/− LSK cells in vivo and their colony-forming capacity in vitro after pharmacologic increase of ROS. (A-B) ROS content of AKT1−/−AKT2−/− LSK cells. The histogram (A) represents one experiment, and the bar graph (B) represents the mean ± SEM (n = 5) of the MFI of the DCF-DA–treated cells normalized to the HBSS-only–treated cells (no DCF-DA) from each experiment. LSK cells were sorted from bone marrow chimeras, incubated with DCF-DA, and analyzed by flow cytometry. (C-D) ROS content in DN3 thymocytes. Thymocytes from 16-week primary competitive bone marrow chimeras were surface stained with CD4, CD8, CD25, and CD45.2. DN3 staged thymocytes were identified by CD4−CD8−CD25+ phenotype. The histogram (C) represents one experiment, and the bar graph (D) represents the mean ± SEM (n = 3) of the DCF-DA MFI of CD45.2+ cells divided by the DCF-DA MFI of CD45.1+ cells in the same tube. (E) Colony-forming capacity of LSK cells treated with BSO. The graphs represent the total number of colonies found in each culture as a percentage of those generated in the absence of BSO (no BSO = 1) and represent the mean ± SEM (n = 5). Equal numbers of freshly sorted LSK cells were plated in methylcellulose cultures with increasing concentrations of BSO for 10 days before analysis. The number of colonies per plate (mean ± SEM) that used AKT1+/+AKT2+/− cells was 0μM BSO 45 ± 3, 0.0167μM BSO 34 ± 3, 0.020μM BSO 26 ± 2, 0.025μM BSO 29 ± 1, 0.050μM BSO 15 ± 2, and 0.100μM BSO no growth and for AKT1−/−AKT2−/− cells was 0μM BSO 32 ± 2, 0.0167μM BSO 38 ± 1, 0.020μM BSO 32 ± 3, 0.025μM BSO 39 ± 3, 0.050μM BSO 23 ± 2, and 0.100μM BSO 3 ± 1. (F) Effect of BSO treatment on LTC-ICs. LSK cells were grown in LTC-IC cultures for 2 weeks in the presence of 0.025μM BSO. Then, equal numbers of cells were harvested from the LTC-IC cultures and plated in methylcellulose in the absence of BSO for 10 days. The graphs represent the total number of colonies found in each methylcellulose culture as a percentage of those generated in the absence of BSO (no BSO = 1) and represent the mean ± SEM (n = 3).

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