Figure 6
Role of Rac in the induction of mitochondrial ROS in TKI-resistant BCR-ABL1 cells and CD34+ CML-CP cells treated with imatinib. (A) Rac activation was examined in 32Dcl3 parental cells (P) and clones expressing nonmutated BCR-ABL1 (nm) and the indicated TKI-resistant mutants (Y253H, T315I, and H396P) using a PAK-binding domain pull-down assay; top box shows the Rac-GTP protein content, bottom box shows the expression of BCR-ABL1 protein variants. (B) The indicated cells were transfected with HA-Rac(T17N)-IRES-GFP (white bars) or IRES-GFP (gray bars) retroviral particles and mitochondrial ·O2− was measured with MSR in GFP+ cells. (C-E) CD34+ cells from healthy donors (N) and from CML-CP patients were incubated with 1μM imatinib (CML + IM) or placebo (CML) for 12 hours in the presence of growth factors. (C) Tyrosine phosphorylation (P.Tyr) of total cellular proteins was analyzed by Western blot using anti-phosphotyrosine Ab. (D) Top panel is a representative Western blot analysis of the Rac-GTP active form detected using a PAK-binding domain pull-down assay; bottom panel shows the total Rac protein in cell lysates by densitometric analysis of Rac-GTP. (E) Total cellular and mitochondrial ·O2− was measured with the use of DHE and MSR, respectively. Results represent mean values of 2 or 3 measurements/group ± SD. *P < .05 compared with cells transfected with an empty plasmid (B) or normal counterparts (D).

Role of Rac in the induction of mitochondrial ROS in TKI-resistant BCR-ABL1 cells and CD34+ CML-CP cells treated with imatinib. (A) Rac activation was examined in 32Dcl3 parental cells (P) and clones expressing nonmutated BCR-ABL1 (nm) and the indicated TKI-resistant mutants (Y253H, T315I, and H396P) using a PAK-binding domain pull-down assay; top box shows the Rac-GTP protein content, bottom box shows the expression of BCR-ABL1 protein variants. (B) The indicated cells were transfected with HA-Rac(T17N)-IRES-GFP (white bars) or IRES-GFP (gray bars) retroviral particles and mitochondrial ·O2 was measured with MSR in GFP+ cells. (C-E) CD34+ cells from healthy donors (N) and from CML-CP patients were incubated with 1μM imatinib (CML + IM) or placebo (CML) for 12 hours in the presence of growth factors. (C) Tyrosine phosphorylation (P.Tyr) of total cellular proteins was analyzed by Western blot using anti-phosphotyrosine Ab. (D) Top panel is a representative Western blot analysis of the Rac-GTP active form detected using a PAK-binding domain pull-down assay; bottom panel shows the total Rac protein in cell lysates by densitometric analysis of Rac-GTP. (E) Total cellular and mitochondrial ·O2 was measured with the use of DHE and MSR, respectively. Results represent mean values of 2 or 3 measurements/group ± SD. *P < .05 compared with cells transfected with an empty plasmid (B) or normal counterparts (D).

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