Figure 5
Figure 5. Rac2 induces mitochondrial ROS, oxidative DNA damage, and genomic instability. (A) BCR-ABL1-32Dcl3 cells were transfected with pMIG-HA-Rac(T17N)–IRES-GFP or LXSP-Rac(T17N) (T17N) and pMIG-IRES-GFP or LXSP (E) retroviruses. Expression of HA-tagged Rac(T17N) mutant and total Rac was detected by Western blot analysis. Results from cells expressing E and T17N are represented by gray and white bars, respectively. ΔΨm (JC-1 red/green fluorescence ratio), electron transport between MRC complexes I-III and II-III, ROS (MSR and CC1 fluorescence), 8-oxoG, and DSBs (marked by γ-H2AX) were assessed (respective panels). (B) CD34+ CML-CP cells were transfected with HA-Rac(T17N)–IRES-GFP (T17N) or IRES-GFP (E) retroviruses. Western blot analyses of the expression of HA-tagged Rac(T17N) mutant and total Rac in GFP+ cells. ROS (bar panels) were measured with MSR and CC1. CD34+ (C), CD34+CD38− (D), and quiescent CD34+CTVmax (E) CML-CP cells were untreated (−) or treated with NSC23766 or EHT1864. (C) Western blot. Rac activation assay: reaction samples (topbox) and total cell lysates (bottom box) were analyzed for Rac-GTP and total Rac protein content, respectively. (C-E bar panels) ROS (MRC and DCFDA fluorescence), 8-oxoG, and γ-H2AX were measured in untreated cells (gray bars) and in cells treated with NSC23766 (white bars) and EHT1864 (striped bars). (F) BCR-ABL1 detected by Western analysis in double-positive, Rac1Δ/Δ, Rac2−/−, Rac3−/−, and Rac1Δ/ΔRac2−/− muBMCs. ROS (MSR and CC1 fluorescence) and oxidative DNA lesions (8-oxoG) and DSBs (γ-H2AX) were detected (bar panels). (G) BCR-ABL1-32Dcl3 cells were transfected with pGFP-V-RS retroviral vector encoding Rac2-specific shRNA (shRNA) or scrambled RNA (Scr). Down-regulation of Rac2 in GFP+ cells was detected by Western blot analysis. ROS (MRC and CC1 fluorescence) were measured in GFP+ cells expressing Scr (gray bars) and shRNA (white bars). Results represent mean values of a minimum of 3 measurements/group ± SD (A-E,G) and mean values of 3-14 muBMC samples/group ± SD (F). *P < .05 compared with empty/Scr plasmid (A,B,G), untreated cells (C-E), and double-positive cells (F).

Rac2 induces mitochondrial ROS, oxidative DNA damage, and genomic instability. (A) BCR-ABL1-32Dcl3 cells were transfected with pMIG-HA-Rac(T17N)–IRES-GFP or LXSP-Rac(T17N) (T17N) and pMIG-IRES-GFP or LXSP (E) retroviruses. Expression of HA-tagged Rac(T17N) mutant and total Rac was detected by Western blot analysis. Results from cells expressing E and T17N are represented by gray and white bars, respectively. ΔΨm (JC-1 red/green fluorescence ratio), electron transport between MRC complexes I-III and II-III, ROS (MSR and CC1 fluorescence), 8-oxoG, and DSBs (marked by γ-H2AX) were assessed (respective panels). (B) CD34+ CML-CP cells were transfected with HA-Rac(T17N)–IRES-GFP (T17N) or IRES-GFP (E) retroviruses. Western blot analyses of the expression of HA-tagged Rac(T17N) mutant and total Rac in GFP+ cells. ROS (bar panels) were measured with MSR and CC1. CD34+ (C), CD34+CD38− (D), and quiescent CD34+CTVmax (E) CML-CP cells were untreated (−) or treated with NSC23766 or EHT1864. (C) Western blot. Rac activation assay: reaction samples (topbox) and total cell lysates (bottom box) were analyzed for Rac-GTP and total Rac protein content, respectively. (C-E bar panels) ROS (MRC and DCFDA fluorescence), 8-oxoG, and γ-H2AX were measured in untreated cells (gray bars) and in cells treated with NSC23766 (white bars) and EHT1864 (striped bars). (F) BCR-ABL1 detected by Western analysis in double-positive, Rac1Δ/Δ, Rac2−/−, Rac3−/−, and Rac1Δ/ΔRac2−/− muBMCs. ROS (MSR and CC1 fluorescence) and oxidative DNA lesions (8-oxoG) and DSBs (γ-H2AX) were detected (bar panels). (G) BCR-ABL1-32Dcl3 cells were transfected with pGFP-V-RS retroviral vector encoding Rac2-specific shRNA (shRNA) or scrambled RNA (Scr). Down-regulation of Rac2 in GFP+ cells was detected by Western blot analysis. ROS (MRC and CC1 fluorescence) were measured in GFP+ cells expressing Scr (gray bars) and shRNA (white bars). Results represent mean values of a minimum of 3 measurements/group ± SD (A-E,G) and mean values of 3-14 muBMC samples/group ± SD (F). *P < .05 compared with empty/Scr plasmid (A,B,G), untreated cells (C-E), and double-positive cells (F).

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