Supplementary Material for: Type 2 Diabetes Promotes Cell Centrosome Amplification via AKT-ROS-Dependent Signalling of ROCK1 and 14-3-3σ
2018-05-16T13:29:15Z (GMT) by
<b><i>Background/Aims:</i></b> Type 2 diabetes is associated with oxidative stress and DNA damage which can cause centrosome amplification. Thus, the study investigated centrosome amplification in type 2 diabetes and the underlying mechanisms. <b><i>Methods:</i></b> Centrosome numbers in human peripheral blood mononuclear blood cells (PBMC) from healthy subjects and patients with type 2 diabetes were compared to access the association between type 2 diabetes and centrosome amplification. Colon cancer cells were used to investigate the molecular mechanisms underlying the centrosome amplification triggered by high glucose, insulin and palmitic acid. Western blot analysis was used to quantify the level of protein and protein phosphorylation. Immunofluorescent staining was performed to detect centrosomes. ROS was quantified using flow cytometry technique. Transcriptpmic profiling was performed using Illumina HiSeq<sup>TM</sup>500 platform. <b><i>Results:</i></b> We found that centrosome amplification was increased PBMC from the type 2 diabetic patients, which correlated with the levels of fasting blood glucose and HbA1c. High glucose, insulin and palmitic acid, alone or in combinations, induced ROS production and centrosome amplification. Together, they increased AKT activation as well as the expression, binding and centrosome translation of ROCK1 and 14-3-3σ. Results from further analyses showed that AKT-ROS-dependent upregulations of expression, binding and centrosome translocation of ROCK1 and 14-3-3σ was the molecular pathway underlying the centrosome amplification <i>in vitro</i> triggered by high glucose, insulin and palmitic acid. Moreover, the key <i>in vitro</i> molecular signalling events activated by high glucose, insulin and palmitic acid were verified in PBMC from the patients with type 2 diabetes. <b><i>Conclusion:</i></b> Our results show that type 2 diabetes promotes cell centrosome amplification, and suggest that the diabetic pathophysiological factors-activated AKT-ROS-dependent signalling of ROCK1 and 14-3-3σ is the underlying molecular mechanism.