1. Busulfan Triggers Intrinsic Mitochondrial-Dependent Platelet Apoptosis Independent of Platelet Activation 
Jianlin Qiao, Yulu Wu, Yun Liu, Xiaoqian Li, Xiaoqing Wu, Na Liu, Feng Zhu, Kunming Qi, Hai Cheng, Depeng Li, Hongchun Li, Zhenyu Li, Lingyu Zeng, Ping Ma, Kailin Xu 
Biology of Blood and Marrow Transplantation 
Volume 22, Issue 9, Pages (September 2016)
DOI: /j.bbmt
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

2. Figure 1
Measurement of mitochondrial membrane depolarization in busulfan-treated platelets. Isolated platelets were incubated with different concentrations of busulfan followed by measurement of mitochondrial membrane depolarization using MitoProbe JC-1 Assay Kit according to manufacturer's instructions by flow cytometry. Equal amount of DMSO was served as a vehicle and carbonyl cyanide m-chlorophenyl hydrazine (CCCP) was used as a positive control. Compared with vehicle, **P < .01.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

3. Figure 2
Analysis of platelet activation after busulfan treatment. After incubation with different concentrations of busulfan, isolated platelets were stained with PE-conjugated antihuman/mouse P-selectin (A) or FITC-conjugated PAC-1 antibody (B) and then analyzed by flow cytometry. An equal amount of DMSO was served as a vehicle and collagen (10 µg/mL) was used as a positive control.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

4. Figure 3
Western blot analysis of platelet apoptosis. Whole proteins were isolated from platelets after busulfan treatment and separated on 10% SDS-PAGE, followed by transferring to an NC membrane. The membranes were then blotted with antibody against Bcl-2, Bax, or caspase-3. Bound antibody was visualized using HRP-conjugated secondary antibody and enhanced chemiluminescence (A). Intensity of positive band was quantified using Image J software. Expression of Bcl-2, Bax, and cleaved caspase 3 was quantified as a ratio to β-actin (B-D). Compared to vehicle, *P < .05, **P < .01.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

5. Figure 4
Analysis of Q-VD-Oph' effect on platelet apoptosis. Whole proteins were extracted from isolated platelets after busulfan (1.5 mg/mL) treatment in the presence or absence of caspase inhibitor Q-VD-Oph (50 µM), separated on 10% SDS-PAGE, and then transferred to an NC membrane. The membranes were blotted with antibody against Bcl-2, Bax, or caspase-3 followed by band visualization using HRP-conjugated secondary antibody and enhanced chemiluminescence. Intensity of positive band was quantified using Image J software. Expression of Bcl-2, Bax and cleaved caspase 3 was quantified as a ratio to β-actin (B-D). Compared to vehicle, *P < .05, **P < .01, ns: not significant.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

6. Figure 5
Platelet aggregation in response to collagen or ADP. Platelet rich plasma was isolated from citrate-anticoagulated venous blood and incubated with busulfan (1 mg/mL) or an equal amount of DMSO (vehicle) in a 37°C water bar for 1 hour followed by measurement of platelet aggregation in response to collagen (2.5 µg/ml) or ADP (5 µM) in Helena Aggram. Maximum platelet aggregation (%) was recorded for the comparison between vehicle and busulfan. *P < .05.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

7. Figure 6
Surface expression of platelet receptor GPIbα, GPVI, and <alpha>IIb<beta>3. After busulfan treatment, platelets were stained with FITC-conjugated anti-CD42b antibody, antihuman glycoprotein VI purified antibody (followed by addition of FITC-conjugated goat antimouse IgG) and FITC-conjugated mouse antihuman CD41a antibody for measurement of the expression of these surface receptors by flow cytometry. The expression was represented as geometric mean (Geo Mean).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

8. Figure 7
Peripheral circulating platelet count after busulfan administration. 20 mg/kg of busulfan was intraperitoneally administrated into each mouse (n = 3) followed by measuring peripheral circulating platelet counts at indicated time points (A). Additionally, 10 mg/kg of QVD-Oph was injected into mice after busulfan administration and platelet count was measured 12 hours after busulfan injection (B). *P < .05.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions