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Figure 4. Deferasirox exposure increases ROS levels. KG-1a cells were separated into CD34+CD38−and CD34+CD38+ fractions (A−G). CD34+CD38−CD123+ and CD34+CD38+CD123+ cells were isolated from newly diagnosed AML patients (n = 3) (A−D). All fractions were cul- tured with 5 mmol/L or 20 mmol/L deferasirox for the indicated times. (A) Representative flow cytometric analysis of ROS production using H2DCFDA staining in KG-1a and AML patient (specimen AML053) cell fractions exposed to deferasirox for 3 hours. (B) Relative ROS levels calculated as fold change of median fluorescence intensity over unstained control § SEM (n = 3, patient samples: AML33, AML51, and AML53). (C) Representative flow cytometric overlays for mBCl fluorescence from KG-1a and AML patient (specimen AML057) cell fractions exposed to 5 mmol/L or 20 mmol/L deferasirox for 30 min or to 3 hours of 800 mmol/L NAC pretreatment + 30 min of 20 mmol/L deferasirox. (D) Relative free thiol groups levels as the fold change of median mBCl fluorescence intensity over unstained control § SEM (n = 3, patient samples: AML54, AML56, and AML57). (E) Representative flow cytometric overlays for MitoSOX fluorescence of CD34+CD38−and CD34+CD38+ KG-1a cells exposed to 5 mmol/L or 20 mmol/L deferasirox for 30 min. Exposure to 1−10 mmol/L <t>doxorubicin</t> for 18 hours was used as a positive control. (F) Relative superoxide levels as the fold change of median MitoSox fluorescence intensity over unstained control § SEM (n = 3). (G) CD34+CD38−and CD34+CD38+ KG-1a cells were exposed to 20 mmol/L deferasirox for 48 hours or pretreated with 400 mmol/L or 800 mmol/L NAC for 1 hour, then washed and treated with 20 mmol/L deferasirox for 48 hours or exposed to 50-100 mmol/L BSO for 48 hours. Apoptosis was evaluated by flow cytometry using Annexin V and 7-AAD. Percentages of apoptotic cells (Annexin V+) are pre- sented § SEM (n = 3). One-way ANOVA followed by Bonferroni’s post hoc test were used to compare the different treatments. *p < 0.05, **p < 0.01.
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Figure 4. Deferasirox exposure increases ROS levels. KG-1a cells were separated into CD34+CD38−and CD34+CD38+ fractions (A−G). CD34+CD38−CD123+ and CD34+CD38+CD123+ cells were isolated from newly diagnosed AML patients (n = 3) (A−D). All fractions were cul- tured with 5 mmol/L or 20 mmol/L deferasirox for the indicated times. (A) Representative flow cytometric analysis of ROS production using H2DCFDA staining in KG-1a and AML patient (specimen AML053) cell fractions exposed to deferasirox for 3 hours. (B) Relative ROS levels calculated as fold change of median fluorescence intensity over unstained control § SEM (n = 3, patient samples: AML33, AML51, and AML53). (C) Representative flow cytometric overlays for mBCl fluorescence from KG-1a and AML patient (specimen AML057) cell fractions exposed to 5 mmol/L or 20 mmol/L deferasirox for 30 min or to 3 hours of 800 mmol/L NAC pretreatment + 30 min of 20 mmol/L deferasirox. (D) Relative free thiol groups levels as the fold change of median mBCl fluorescence intensity over unstained control § SEM (n = 3, patient samples: AML54, AML56, and AML57). (E) Representative flow cytometric overlays for MitoSOX fluorescence of CD34+CD38−and CD34+CD38+ KG-1a cells exposed to 5 mmol/L or 20 mmol/L deferasirox for 30 min. Exposure to 1−10 mmol/L <t>doxorubicin</t> for 18 hours was used as a positive control. (F) Relative superoxide levels as the fold change of median MitoSox fluorescence intensity over unstained control § SEM (n = 3). (G) CD34+CD38−and CD34+CD38+ KG-1a cells were exposed to 20 mmol/L deferasirox for 48 hours or pretreated with 400 mmol/L or 800 mmol/L NAC for 1 hour, then washed and treated with 20 mmol/L deferasirox for 48 hours or exposed to 50-100 mmol/L BSO for 48 hours. Apoptosis was evaluated by flow cytometry using Annexin V and 7-AAD. Percentages of apoptotic cells (Annexin V+) are pre- sented § SEM (n = 3). One-way ANOVA followed by Bonferroni’s post hoc test were used to compare the different treatments. *p < 0.05, **p < 0.01.
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Figure 4. Deferasirox exposure increases ROS levels. KG-1a cells were separated into CD34+CD38−and CD34+CD38+ fractions (A−G). CD34+CD38−CD123+ and CD34+CD38+CD123+ cells were isolated from newly diagnosed AML patients (n = 3) (A−D). All fractions were cul- tured with 5 mmol/L or 20 mmol/L deferasirox for the indicated times. (A) Representative flow cytometric analysis of ROS production using H2DCFDA staining in KG-1a and AML patient (specimen AML053) cell fractions exposed to deferasirox for 3 hours. (B) Relative ROS levels calculated as fold change of median fluorescence intensity over unstained control § SEM (n = 3, patient samples: AML33, AML51, and AML53). (C) Representative flow cytometric overlays for mBCl fluorescence from KG-1a and AML patient (specimen AML057) cell fractions exposed to 5 mmol/L or 20 mmol/L deferasirox for 30 min or to 3 hours of 800 mmol/L NAC pretreatment + 30 min of 20 mmol/L deferasirox. (D) Relative free thiol groups levels as the fold change of median mBCl fluorescence intensity over unstained control § SEM (n = 3, patient samples: AML54, AML56, and AML57). (E) Representative flow cytometric overlays for MitoSOX fluorescence of CD34+CD38−and CD34+CD38+ KG-1a cells exposed to 5 mmol/L or 20 mmol/L deferasirox for 30 min. Exposure to 1−10 mmol/L <t>doxorubicin</t> for 18 hours was used as a positive control. (F) Relative superoxide levels as the fold change of median MitoSox fluorescence intensity over unstained control § SEM (n = 3). (G) CD34+CD38−and CD34+CD38+ KG-1a cells were exposed to 20 mmol/L deferasirox for 48 hours or pretreated with 400 mmol/L or 800 mmol/L NAC for 1 hour, then washed and treated with 20 mmol/L deferasirox for 48 hours or exposed to 50-100 mmol/L BSO for 48 hours. Apoptosis was evaluated by flow cytometry using Annexin V and 7-AAD. Percentages of apoptotic cells (Annexin V+) are pre- sented § SEM (n = 3). One-way ANOVA followed by Bonferroni’s post hoc test were used to compare the different treatments. *p < 0.05, **p < 0.01.
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Figure 4. Deferasirox exposure increases ROS levels. KG-1a cells were separated into CD34+CD38−and CD34+CD38+ fractions (A−G). CD34+CD38−CD123+ and CD34+CD38+CD123+ cells were isolated from newly diagnosed AML patients (n = 3) (A−D). All fractions were cul- tured with 5 mmol/L or 20 mmol/L deferasirox for the indicated times. (A) Representative flow cytometric analysis of ROS production using H2DCFDA staining in KG-1a and AML patient (specimen AML053) cell fractions exposed to deferasirox for 3 hours. (B) Relative ROS levels calculated as fold change of median fluorescence intensity over unstained control § SEM (n = 3, patient samples: AML33, AML51, and AML53). (C) Representative flow cytometric overlays for mBCl fluorescence from KG-1a and AML patient (specimen AML057) cell fractions exposed to 5 mmol/L or 20 mmol/L deferasirox for 30 min or to 3 hours of 800 mmol/L NAC pretreatment + 30 min of 20 mmol/L deferasirox. (D) Relative free thiol groups levels as the fold change of median mBCl fluorescence intensity over unstained control § SEM (n = 3, patient samples: AML54, AML56, and AML57). (E) Representative flow cytometric overlays for MitoSOX fluorescence of CD34+CD38−and CD34+CD38+ KG-1a cells exposed to 5 mmol/L or 20 mmol/L deferasirox for 30 min. Exposure to 1−10 mmol/L <t>doxorubicin</t> for 18 hours was used as a positive control. (F) Relative superoxide levels as the fold change of median MitoSox fluorescence intensity over unstained control § SEM (n = 3). (G) CD34+CD38−and CD34+CD38+ KG-1a cells were exposed to 20 mmol/L deferasirox for 48 hours or pretreated with 400 mmol/L or 800 mmol/L NAC for 1 hour, then washed and treated with 20 mmol/L deferasirox for 48 hours or exposed to 50-100 mmol/L BSO for 48 hours. Apoptosis was evaluated by flow cytometry using Annexin V and 7-AAD. Percentages of apoptotic cells (Annexin V+) are pre- sented § SEM (n = 3). One-way ANOVA followed by Bonferroni’s post hoc test were used to compare the different treatments. *p < 0.05, **p < 0.01.
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Image Search Results


Figure 4. Deferasirox exposure increases ROS levels. KG-1a cells were separated into CD34+CD38−and CD34+CD38+ fractions (A−G). CD34+CD38−CD123+ and CD34+CD38+CD123+ cells were isolated from newly diagnosed AML patients (n = 3) (A−D). All fractions were cul- tured with 5 mmol/L or 20 mmol/L deferasirox for the indicated times. (A) Representative flow cytometric analysis of ROS production using H2DCFDA staining in KG-1a and AML patient (specimen AML053) cell fractions exposed to deferasirox for 3 hours. (B) Relative ROS levels calculated as fold change of median fluorescence intensity over unstained control § SEM (n = 3, patient samples: AML33, AML51, and AML53). (C) Representative flow cytometric overlays for mBCl fluorescence from KG-1a and AML patient (specimen AML057) cell fractions exposed to 5 mmol/L or 20 mmol/L deferasirox for 30 min or to 3 hours of 800 mmol/L NAC pretreatment + 30 min of 20 mmol/L deferasirox. (D) Relative free thiol groups levels as the fold change of median mBCl fluorescence intensity over unstained control § SEM (n = 3, patient samples: AML54, AML56, and AML57). (E) Representative flow cytometric overlays for MitoSOX fluorescence of CD34+CD38−and CD34+CD38+ KG-1a cells exposed to 5 mmol/L or 20 mmol/L deferasirox for 30 min. Exposure to 1−10 mmol/L doxorubicin for 18 hours was used as a positive control. (F) Relative superoxide levels as the fold change of median MitoSox fluorescence intensity over unstained control § SEM (n = 3). (G) CD34+CD38−and CD34+CD38+ KG-1a cells were exposed to 20 mmol/L deferasirox for 48 hours or pretreated with 400 mmol/L or 800 mmol/L NAC for 1 hour, then washed and treated with 20 mmol/L deferasirox for 48 hours or exposed to 50-100 mmol/L BSO for 48 hours. Apoptosis was evaluated by flow cytometry using Annexin V and 7-AAD. Percentages of apoptotic cells (Annexin V+) are pre- sented § SEM (n = 3). One-way ANOVA followed by Bonferroni’s post hoc test were used to compare the different treatments. *p < 0.05, **p < 0.01.

Journal: Experimental hematology

Article Title: Deferasirox selectively induces cell death in the clinically relevant population of leukemic CD34 + CD38 - cells through iron chelation, induction of ROS, and inhibition of HIF1α expression.

doi: 10.1016/j.exphem.2018.10.010

Figure Lengend Snippet: Figure 4. Deferasirox exposure increases ROS levels. KG-1a cells were separated into CD34+CD38−and CD34+CD38+ fractions (A−G). CD34+CD38−CD123+ and CD34+CD38+CD123+ cells were isolated from newly diagnosed AML patients (n = 3) (A−D). All fractions were cul- tured with 5 mmol/L or 20 mmol/L deferasirox for the indicated times. (A) Representative flow cytometric analysis of ROS production using H2DCFDA staining in KG-1a and AML patient (specimen AML053) cell fractions exposed to deferasirox for 3 hours. (B) Relative ROS levels calculated as fold change of median fluorescence intensity over unstained control § SEM (n = 3, patient samples: AML33, AML51, and AML53). (C) Representative flow cytometric overlays for mBCl fluorescence from KG-1a and AML patient (specimen AML057) cell fractions exposed to 5 mmol/L or 20 mmol/L deferasirox for 30 min or to 3 hours of 800 mmol/L NAC pretreatment + 30 min of 20 mmol/L deferasirox. (D) Relative free thiol groups levels as the fold change of median mBCl fluorescence intensity over unstained control § SEM (n = 3, patient samples: AML54, AML56, and AML57). (E) Representative flow cytometric overlays for MitoSOX fluorescence of CD34+CD38−and CD34+CD38+ KG-1a cells exposed to 5 mmol/L or 20 mmol/L deferasirox for 30 min. Exposure to 1−10 mmol/L doxorubicin for 18 hours was used as a positive control. (F) Relative superoxide levels as the fold change of median MitoSox fluorescence intensity over unstained control § SEM (n = 3). (G) CD34+CD38−and CD34+CD38+ KG-1a cells were exposed to 20 mmol/L deferasirox for 48 hours or pretreated with 400 mmol/L or 800 mmol/L NAC for 1 hour, then washed and treated with 20 mmol/L deferasirox for 48 hours or exposed to 50-100 mmol/L BSO for 48 hours. Apoptosis was evaluated by flow cytometry using Annexin V and 7-AAD. Percentages of apoptotic cells (Annexin V+) are pre- sented § SEM (n = 3). One-way ANOVA followed by Bonferroni’s post hoc test were used to compare the different treatments. *p < 0.05, **p < 0.01.

Article Snippet: Exposure to 1 and 10mM doxorubicin (DOX) (Selleckchem) for 18 hours was used as positive control.

Techniques: Isolation, Staining, Control, Positive Control, Cytometry