Fig 1.
Akt3 depletion results in micronuclei formation.
A) ECs were subjected to Akt3 depletion or scrambled control. Fixed cells were assessed for nuclear structure using immunofluorescence with antibodies directed against Lamin A/C and actin stained with phalloidin. DAPI is used as a nuclear stain. Arrows point to multinuclei and blebbing. 60X images are shown B) Quantitation of micronuclei (MN) under conditions of scrambled control, or Akt1 or Akt3 depletion. Standard deviation and p values are shown. C) Confirmation of Akt1 and Akt3 knockdown by immunoblot analysis. The PI3 kinase p85 subunit is shown as a loading control.
Fig 2.
Akt3 controls the expression of WDR12, a chromosome binding protein.
A) ECs were transfected with an RNAi directed against scrambled control or Akt3 and assessed for expression of WDR12 by Immunoblot analysis. p85 is shown as a loading control. B) Visualization of micronuclei (MN) in response to WDR12 depletion. C) Percent micronuclei was measured in ECs transfected with scrambled control, or RNAi directed against Akt3 or WDR12. Standard deviation is shown. * p < 0.05, **p < 0.05 D) Quantitation of dysmorphic nuclei resulting from overexpression of WDR12 in Akt3 depleted cells.
Fig 3.
WDR12 associates with Aurora B.
A) Immunofluorescence using antibodies against αtubulin, and WDR12 showing WDR12 localization during different stages of mitosis. DAPI is the nuclear stain. B) Immunoprecipitation of Aurora B or WDR12 followed by immunoblot of WDR12 or Aurora B, respectively. C) Co-immunoprecipitation ECs transfected with GFP-tagged Aurora B plus Myc-tagged WDR12 using an antibody directed against GFP followed by immunoblot with anti-Myc. D) RNAi directed against Akt3 or scrambled control and assessed for Aurora B expression by immunoblot. p85 is shown as the loading control.
Fig 4.
Pharmacological induction of mitochondrial dysfunction results in micronuclei and nuclear blebbing.
A) ECs treated with paraquat (1 mM) for 24hr and subjected to immunofluorescence with antibodies directed against lamin A/C, stained with phalloidin and DAPI. Arrows indicate micronuclei and nuclear blebbing. B) Quantitation of micronuclei (MN) in ECs treated with paraquat (1 mM) for 12 or 24hrs. Standard deviations are shown. * p < 0.001, **p < 0,001. C) Mitosox staining of cells treated with paraquat as in A.
Fig 5.
ROS reduces the expression of Akt3, WDR12 and Aurora B leading to micronuclei.
A) Paraquat (1 mM) treatment of ECs in a time course analysis and tested for expression of Akt3, WDR12, and Aurora B by immunoblot. Actin is shown as a loading control. B) ECs treated with paraquat plus and minus MitoQ at the concentrations indicated and assessed for Akt3 and aurora B expression by immunoblot. Actin is shown as a loading control. C) Quantitation of percent mitotic catastrophe in ECs treated with paraquat plus and minus the anti-oxidant N-acetyl glucosamine for 24 hours. * p < 0.001, **p < 0,001 Standard deviations are shown.
Fig 6.
Mitotic catastrophe is dependent on CRM-1 dysregulation.
A) EC treated with paraquat (1 mM) for 24hr plus or minus leptomycin B (50 ng/ml) followed by immunoblot for Aurora B. Vinculin is shown as a loading control. B) EC transfected with vector alone or a CRM-1 expression vector assessed for Aurora B expression. Vinculin is shown as a loading control. C) Paraquat treated cells were treated with TAME (50 or 100mM) and assessed for the expression of Aurora B by immunoblot. Actin is shown as an internal control. D) EC subjected to CRM-1 overexpression and assessed for micronuclei using lamin A/C, αtubulin and DAPI staining. E) Quantitation of mitotic catastrophe in CRM-1 overexpressing cells. p < .01.
Fig 7.
Morphometric analysis of wild type and Akt3 null hearts.
A) H&E staining of wild type versus Akt3 hearts. Top panel indicated areas of measurement (length and width). Bottom panel shows areas measured for compact layers. Quantitation is shown in B) length and width and C) thickness of compact layer. n = 2 wild type and n = 4 Akt3 nulls.
Fig 8.
Akt3 null animals have reduced Aurora B expression and proliferation and increased apoptosis.
Paraffin sections of Akt3 null hearts were subjected to immunofluorescence using antibodies against A) Aurora B, B) Ki67 and C) cleaved caspase 3. Quantitation is shown next to each figure. D) H&E of wild type and Akt3 null hearts. E) Immunoblot of CRM-1, Aurora B in whole heart lysates of wild type and Akt3 null animals. Vinculin is shown as a loading control.
Fig 9.
Model of the role of increased ROS in chromosome missegregation.
Increased ROS results in a reduction in Akt3 expression. This causes an increased expression of CRM-1 and an APC/c dependent turnover of Aurora B kinase.