Ultra thin sections were doubly stained with uranyl acetate and observed under an electron microscope. Statistical analysis Continuous data are presented as mean averages Navitoclax mw with standard deviations. Comparison of continuous data was performed by the Students T test or the Mann Whitney U test using SPSS for WINDOWS, version 12. 0. A p value of less than 0. 05 was considered significant. Results Establishment of NIH 3T3 cells overexpressing functional IRS 1 We chose NIH 3T3 cells as an experimental model to in vestigate the role of IRS 1 in oxidative stress mediated autophagy and cell death. Western blotting confirmed the presence of IRS 1 in wild type NIH 3T3 cells. To mimic the increased expression levels of IRS 1 seen in tumor cells, we established NIH 3T3 cells with stable overexpression of IRS 1.

The levels of total IRS 1 in both the control NIH 3T3 cells and NIH 3T3 cells overexpressing IRS 1 were checked by Western blot analysis. The amount of total IRS 1 was greater in cells infected with retrovirus encoding for the IRS 1 gene than it was in the control cells, indicating that ex ogenous IRS 1 was expressed in abundant quantities. Next, we checked if the expressed IRS 1 was functional by determining whether the well established downstream IRS 1 effectors, including p70 ribosomal protein S6 kin ase, Akt, and ERK were affected by the overex pression of IRS 1. The extent of phosphorylation of p70 S6K at Thr 389, and S6 proteins at Ser 240 244 was greater in cells over expressing IRS 1 than in the control cells treated with or without insulin.

Following insulin treatment, the extent of phosphorylation of Akt at Thr 308 and Ser 473, and the extent of glycogen synthesis kinase 3 beta at Ser 9, was greater in the IRS 1 overexpressing cells than it was in the control cells. In the absence of insulin treatment, there were no obvious differences in the extent of phosphorylation of target proteins between the two groups of cells. The extent of phosphorylation of ERK1 and ERK2 at Thr 202 and Tyr 204 was also greater in cells overexpressing IRS 1 than it was in the control cells under a steady state growth phase. Thus, we successfully established NIH 3T3 cells with stable over expression of functional IRS 1 proteins. Effect of IRS 1 overexpression on basal autophagy IRS 1 increases the activity of class I PI3K Akt signaling and mTOR, which is located downstream of the class I PI3K Akt signaling pathway, and is the core nega tive regulator of autophagy.

Thus, it is possible that autophagy is inhibited in NIH 3T3 cells Dacomitinib that overexpress IRS 1. To confirm this hypothesis, we investigated basal autophagy by following the conversion of LC3B, from LC3B I, which is found in the cytosol as a free form, to LC3B II via conjugation with phosphatidylethanolamine. LC3B II associates with autophagosome membranes, and its generation is a promising autophagosomal mar ker, the amount of LC3 II usually correlates well with the number of autophagosomes.