These results suggest that IL 1b could not induce autophagy in AF cells cultured with 10% FBS. In the second set of experiments, all AF cells were cultured in the find FAQ serum free media. There was no signifi cant increase in the autophagy incidence AF cells cul tured for six hours under serum starvation conditions, whereas there was a significant increase Inhibitors,Modulators,Libraries in both autop hagy and apoptosis incidence of cells after 24 hour serum starvation. Thus, we examined autophagy inci dence in rat AF cells after 12 hours of serum starvation. The results showed that the 12 hour serum deprivation resulted autophagy in around 13% of the rat AF cells. Adding 10 ng ml or more of IL 1b significantly aug mented the autophagy incidence AF cells as quantified with flow cytometry. At the concen tration of 10 ng ml, IL 1b induced an increase of 1.
2 fold of the autophagy incidence. The concentration of 20 ng ml and 50 ng ml induced an increase of 1. 38 and 1. 85 fold of autophagy incidence in AF cellls. The results showed that autophagy incidence was gradually increased over time but IL 1b did not induce autophagy when the AF Inhibitors,Modulators,Libraries cells were cultured with 10% FBS. In contrast, serum deprivation easily induced autophagy in AF cells. More over, IL 1b upregulated the autophagic effect of serum deprivation in a dose dependent manner. In order to further corroborate the findings in our flow cytometry studies, we next examined lysosome activity and mRNA expression of autophagy related genes. As shown in Figure 3c, the density of Lyso Tracker staining did not change when the AF cells were cultured with 10% FBS.
Inhibitors,Modulators,Libraries How ever, the density of Lyso Tracker was Inhibitors,Modulators,Libraries significantly increased when cells were Inhibitors,Modulators,Libraries serum deprived for 12 hours, compared with that of the cells cultured with 10% FBS. Based upon the results of the preliminary study, IL b at the concentration of 20 ng ml was chosen to examine mRNA expression of Beclin 1, Bcl 2, and LC3. Consis tent with the quantification of the rate of autophagy, serum deprivation induced a significant increase in Beclin 1, Bcl 2, and LC3 expression in AF cells, which was not observed over time with serum supplementation. These results suggests that IL 1b is not cap able of inducing autophagy in AF cells by itself, but it can significantly potentiate autophagy under serum star vation at least.
The autophagy in AF cells is partially rescued by 10% FBS treatment To determine whether autophagy could be rescued, we evaluated the impact of nutrient supplementation on the fate of autophagy in AF cells. The AF cells were Crizotinib structure first cultured in serum withdrawal media with IL 1b at the concentration of 10 ng ml for 24 hours to induce autop hagy. According to the results of our preliminary experi ment, re feeding the cells with 10% FBS for three hours significantly reduced the autophagy incidence and in turn led to an increase in the total number of viable cells 12 hours later.