Figure 4
MRC-cIII is responsible for ROS-induced oxidative DNA damage in primitive CML-CP CD34+ subsets and in PV and AML cells. CD34+ (A), CD34+CD38− (B), and quiescent CD34+CTVmax (C) cells from CML-CP patients were incubated without (gray bars) or with (white bars) the indicated inhibitors (R indicates rotenone; Ma, malonate; S, stigmatellin; My, myxothiazol; A, antimycin; and K, KCN) for 3 hours in 17% or 1% O2 (A) and in 17% O2 (B-C). ROS were measured by DCFDA. (D) ROS were also measured by DCFDA in primary FLT3(ITD)–positive AML cells and in JAK2(V617F)–positive PV cells untreated (−) or treated with myxothiazol (My) or stigmatellin (S). Results show relative ROS levels compared with untreated cells. (E) Control L929 cells (E9) and clones containing a defective mutation in complex I (FG23-1) and complex III (A22) were cotransfected with BCR-ABL1–IRES-GFP or IRES-GFP expression plasmid. ROS was measured 72 hours later in GFP+ cells with CC1. Results represent the relative increase of ROS in BCR-ABL1+ cells. 8-oxoG (F) and γ-H2AX (G) DNA lesions were detected in CD34+ CML-CP cells incubated with malonate, antimycin A, or diluent (−) for 48 hours. Results represent mean values of a minimum of 3 measurements/group ± SD. *P < .05 compared with the untreated cells (A-D,F-G) and with E9 (E).

MRC-cIII is responsible for ROS-induced oxidative DNA damage in primitive CML-CP CD34+ subsets and in PV and AML cells. CD34+ (A), CD34+CD38 (B), and quiescent CD34+CTVmax (C) cells from CML-CP patients were incubated without (gray bars) or with (white bars) the indicated inhibitors (R indicates rotenone; Ma, malonate; S, stigmatellin; My, myxothiazol; A, antimycin; and K, KCN) for 3 hours in 17% or 1% O2 (A) and in 17% O2 (B-C). ROS were measured by DCFDA. (D) ROS were also measured by DCFDA in primary FLT3(ITD)–positive AML cells and in JAK2(V617F)–positive PV cells untreated (−) or treated with myxothiazol (My) or stigmatellin (S). Results show relative ROS levels compared with untreated cells. (E) Control L929 cells (E9) and clones containing a defective mutation in complex I (FG23-1) and complex III (A22) were cotransfected with BCR-ABL1–IRES-GFP or IRES-GFP expression plasmid. ROS was measured 72 hours later in GFP+ cells with CC1. Results represent the relative increase of ROS in BCR-ABL1+ cells. 8-oxoG (F) and γ-H2AX (G) DNA lesions were detected in CD34+ CML-CP cells incubated with malonate, antimycin A, or diluent (−) for 48 hours. Results represent mean values of a minimum of 3 measurements/group ± SD. *P < .05 compared with the untreated cells (A-D,F-G) and with E9 (E).

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