Figure 1.
ROCK1 deletion reduces doxorubicin-induced ROS production.
A. Representative images of CM-H2DCFDA staining. WT and ROCK1−/− MEFs were grown on glass coverslips, treated for 4 h with 3 µM doxorubicin and then exposed to 13 µM CM-H2DCFDA. Coverslip was mounted with Vector mounting medium containing DAPI and imaged immediately. Bar, 50 µm. B. Quantitative analysis of the images obtained from fluorescence microscopy with the Leica AF6000 software. Fluorescence levels in WT cells at baseline were arbitrarily set at 1. C and D. Representative results (C) and quantitative analysis (D) of ROS measured by flow cytometry analysis after staining with CM-H2DCFDA. WT and ROCK1−/− cells were treated for 16 h with 3 µM doxorubicin (C) or with the dosages of doxorubicin as indicated (D). The attached cells were stained with 5 µM CM-H2DCFDA, collected and analyzed by flow cytometry in at least three separate experiments. Fluorescence levels in WT cells at baseline were arbitrarily set at 1. *P<0.05 vs. control of the same genotype. #P<0.05 vs. WT under the same treatment condition.
Figure 2.
ROCK1 deletion enhances anti-ROS, pro-survival, anti-apoptotic effects of NAC.
A. ROS levels measured by flow cytometry analysis after CM-H2DCFDA staining of attached WT and ROCK1−/− MEFs after 3 µM doxorubicin and/or 2 mM NAC treatment for 16 h; showing a minimal doxorubicin-induced ROS production in NAC treated ROCK1 deficient MEFs. Fluorescence levels in WT cells at baseline were arbitrarily set at 1. B. MTT assay performed with MEFs treated for 16 h with 3 µM doxorubicin and/or 2 mM NAC showing maximal level of cell viability after doxorubicin treatment in NAC treated ROCK1 deficient MEFs. C. Representative image of Western blot of cleaved caspase-3 and -8 in cell lysates from attached WT and ROCK1 deficient MEFs treated with 3 µM doxorubicin and/or 2 mM NAC for 16 h. Equal amount of proteins were loaded. D. Densitometry analysis of immunoreactive bands of cleaved caspase-3 expressed as percent change relative to WT cells treated with 3 µM doxorubicin, showing a minimal level of caspase activation in NAC treated ROCK1 deficient MEFs. *P<0.05 vs. control of the same genotype. #P<0.05 vs. WT under the same treatment condition. ¶P<0.05 vs. the same genotype under doxorubicin only condition.
Figure 3.
ROCK1 deletion enhances anti-detachment effects of NAC.
A. Representative images of rhodamine-phalloidin staining for F- actin (red), p-MLC staining (green) and DAPI (blue) of WT and ROCK1 deficient cells treated with 3 µM doxorubicin and/or 2 mM NAC for 8 h. Cells showing disruption of central stress fibers are indicated with white arrowheads. ROCK1 deletion, but not NAC treatment, prevents DOX-induced disruption of central stress fibers. Bar, 25 µm. B and C. Representative image (B) of Western blot of p-MLC2 and MLC2, or p-cofilin and cofilin in cell lysates from attached WT and ROCK1−/− MEFs treated with 3 µM doxorubicin and/or 2 mM NAC for 30 min. Quantitative analysis (C) showing reduction of both p-MLC and p-cofilin in WT MEFs after NAC/DOX treatment compared to DOX treatment alone. The ratio of p-MLC to MLC or p-cofilin to cofilin was expressed as fold change relative to WT control. D and E. Both floating and attached cells were collected after treatment for 16 h with 3 µM doxorubicin and/or 2 mM NAC. Floating cell ratio was expressed as percentage of total cells (floating plus attached cells) under each treatment condition (D). Attached cell number was expressed as percentage of attached cells under control condition without treatment (E). *P<0.05 vs. control of the same genotype. #P<0.05 vs. WT under the same treatment condition. ¶P<0.05 vs. the same genotype under doxorubicin only condition.
Figure 4.
ROCK1 deletion reduces NADPH oxidase and Rac1 activity and membrane translocation of p67phox and Rac1 in response to doxorubicin.
NADPH oxidase activity assay (A) and Rac1 activity pull-down assay (B) were performed with WT and ROCK1−/− MEFs after treatment for 16 h with 3 µM doxorubicin showing reduced NADPH oxidase activation in ROCK1 deficient cells. The activity was expressed as fold change relative to WT control. C. Representative image (top) and quantitative analysis (bottom) of Western blot of p67phox and Rac1 performed with membrane fractions from attached WT and ROCK1−/− MEFs treated with 3 µM of doxorubicin at indicated time points. *P<0.05 vs. control of the same genotype. #P<0.05 vs. WT under the same treatment condition.
Figure 5.
ROCK1 deletion enhances the anti-ROS and anti-apoptotic effects of DPI.
A. Representative images of CM-H2DCFDA staining of WT and ROCK1−/− MEFs treated for 4 h with 3 µM doxorubicin and/or 1 µM DPI. Coverslip was mounted with Vector mounting medium containing DAPI and imaged immediately by fluorescence microscopy. Bar, 50 µm. B. Quantitative analysis of the images obtained from fluorescence microscopy with the Leica AF6000 software. Fluorescence level in WT cells under doxorubicin only condition was arbitrarily set at 1. C. Representative image of Western blot of cleaved caspase-3, -8, and -9 in cell lysates from attached WT and ROCK1 deficient MEFs treated for 16 h with 3 µM doxorubicin and/or 1 µM DPI. Equal amount of proteins were loaded. D. Densitometry analysis of immunoreactive bands of cleaved caspase-3 expressed as percent change relative to WT cells treated with 3 µM doxorubicin, showing minimal level of caspase activation in DPI treated ROCK1 deficient MEFs. *P<0.05 vs. control of the same genotype. #P<0.05 vs. WT under the same treatment condition. ¶P<0.05 vs. the same genotype under doxorubicin only condition.
Figure 6.
ROCK1 deletion preserves central stress fibers in DPI and doxorubicin treated cells.
A. Representative images of rhodamine-phalloidin staining for F-actin (red) and DAPI staining (blue) of WT and ROCK1−/− cells treated with 3 µM doxorubicin and/or 1 µM DPI for 16 h. Cells showing disruption of central stress fibers are indicated with white arrowheads. DPI treatment alone induces disruption of central stress fibers in WT cells. ROCK1 deletion prevents doxorubicin and/or DPI-induced disruption of central stress fibers. Bar, 25 µm. B and C. DPI increases doxorubicin-induced cell detachment in WT MEFs but not in ROCK1 deficient MEFs. Floating cells (B) and attached cells (C) were collected at 16 h post-treatment with 3 µM doxorubicin and/or 1 µM DPI. Floating cell ratio was expressed as percentage of total cells (floating plus attached cells) under each treatment condition. Attached cell number was expressed as percentage of attached cells under control condition without treatment. *P<0.05 vs. control of the same genotype. #P<0.05 vs. WT under the same treatment condition. ¶P<0.05 vs. the same genotype under doxorubicin only condition.
Figure 7.
ROCK2 deletion reduces DOX-induced ROS production and caspase activation.
A. Representative images of CM-H2DCFDA staining of WT and ROCK2−/− MEFs treated for 4 h with 3 µM of doxorubicin. Coverslip was mounted with Vector mounting medium containing DAPI and imaged immediately by fluorescence microscopy. Bar, 50 µm. B. Quantitative analysis of the images obtained from fluorescence microscopy with the Leica AF6000 software. Fluorescence levels in WT cells at baseline were arbitrarily set at 1, showing reduced doxorubicin-induced ROS production in ROCK2 deficient cells. C. Representative image of Western blot of ROCK2, cleaved caspase-3, -8, and -9 in cell lysates from attached WT and ROCK2−/− MEFs treated for 16 h with increasing dosages of doxorubicin as indicated. D. Quantitative analysis of immunoreactive bands of cleaved capsase-3 (n = 4–6 for each condition), expressed as percent change relative to WT cells treated with doxorubicin, showing reduced caspase activation in ROCK2 deficient cells treated with 3 or 5 µM doxorubicin. *P<0.05 vs. control of the same genotype. #P<0.05 vs. WT under the same treatment condition.
Figure 8.
ROCK2 deletion does not preserve central stress fibers in doxorubicin-treated cells with or without NAC or DPI, and does not enhance the anti-apoptotic effect of DPI.
A. Representative images of rhodamine-phalloidin staining for F-actin (red) and DAPI staining (blue) of ROCK2−/− cells treated with 3 µM doxorubicin and/or 2 mM NAC or 1 µM DPI for 16 h. Cells showing disruption of central stress fibers are indicated with white arrowheads. Cells showing folded periphery membranes are indicated with white arrowheads. DPI treatment alone induces periphery membrane folding in ROCK2−/− cells. Bar, 50 µm. B and C. Floating and attached cells were collected at 16 h post-treatment with 3 µM doxorubicin and/or 2 mM NAC (B) and/or 1 µM DPI (C). Floating cell ratio was expressed as percentage of total cells (floating plus attached cells) under each treatment condition. No significant difference was observed between WT and ROCK2 deficient cells under the same treatment condition. ROCK2 deletion does not enhance the anti-detachment effects of NAC. DPI treatment increases doxorubicin-induced cell detachment in WT and ROCK2 deficient MEFs. D. Representative image of Western blot of cleaved caspase-3, -8, and -9 in cell lysates from attached WT and ROCK2 deficient MEFs treated for 16 h with 3 µM doxorubicin and/or 1 µM DPI. E. Densitometry analysis of immunoreactive bands of cleaved caspase-3 expressed as percent change relative to WT cells treated with 3 µM doxorubicin, showing no further reduction in caspase activation by DPI treatment in doxorubicin-treated ROCK2 deficient MEFs. *P<0.05 vs. control of the same genotype. #P<0.05 vs. WT under the same treatment condition. ¶P<0.05 vs. the same genotype under doxorubicin only condition.
Figure 9.
The diagram summarizes the effects of anti-oxidants (NAC and DPI) and ROCK1 deficiency in opposing ROS production, apoptosis, actin cytoskeleton reorganization, and cell detachment induced by doxorubicin. NAC reduces ROS production leading to reduced apoptosis. NAC also decreases actin cytoskeleton remodeling in part through decreasing MLC phosphorylation and actomyosin contraction leading to reduced cell detachment. However, NAC inhibits actin polymerization through reducing cofilin phosphorylation, and therefore does not prevent the disruption of central stress fibers. DPI reduces ROS production and apoptosis, supporting a contributory role in doxorubicin-induced ROS production. ROCK1 deficiency reduces ROS production, in part through inhibiting NADPH oxidase activation. Finally, ROCK1 deficiency presents a unique property of preserving central stress fiber stability through reducing MLC phosphorylation and preserving cofilin phosphorylation, leading to the preserved cell adhesion which then enhances the protective effects of antioxidant treatment against doxorubicin-induced ROS production, apoptosis and cell detachment.