For lipid analy sis the results inhibitor Gemcitabine are presented as means with standard devia tion and comparisons were made by ANOVA followed by Tukeys post hoc multi comparisons test. For correlations, Spearmans non parametric test was used. P values of less than 0. 05 were considered statistically significant. Autophagy is the major catabolic pathway for degrada tion of dysfunctional organelles and macromolecules. First characterized in yeast genetically conserved ATG proteins emerged that participate in and regulate the process of autophagy. ATG proteins are grouped into 1 a Class III phosphatidylinositol 3 kinase complex functioning in vesicle nucleation, 2 a serine threonine kinase complex involved in induction of autophagy, and 3 ubiquitin like protein conjugating systems ATG12 and ATG8 that promote maturation of vesicles.

The mammalian homologue of ATG8 is LC3, an interactive partner of microtubule associated protein MAP1A MAP1B and C19ORF5. The LC3 precursor is truncated to LC3I then conjugated with phosphatidylethanolamine to membrane associated LC3II mediated by the ATG5 ATG12 conjugate. The LC3II associated isolation membranes mature and fuse with lysosomes to form autolysosomes in which LC3II is degraded along with the cargo of the autopha gosome. The autophagic process can be divided into autophagosomal biogenesis and autophagosomal degra dation based on the fate of LC3 isoforms. Both LC3I and LC3II are used as markers for autophagy at differ ent steps and levels reveal a balance of biogenesis and conversion degradation, respectively.

Caution is required to interpret the results from immunoblot since the LC3 levels are dynamically altered. Increasing levels of LC3I suggest increased production of LC3I and reduced conversion to LC3II while increasing levels Carfilzomib of LC3II indicate enhanced conversion of LC3I to LC3II and impaired degradation through lysosomes. For example, the accumulation of LC3II in cells cultured in Hanks media has been interpreted as a consequence of autop hagic activation based on the assumption that the capa city of lysosomal degradation remains constant. However, such accumulation could also be caused by an impairment of lysosomal degradation. In order to correctly interpret the LC3 immunoblot data, lysosomal inhibitor NH4Cl or bafilomycin A1 are used to block autophagosomal degradation in lysosomes to show the total amount of converted LC3II during blockade. An increase in the total amount of LC3II in the pre sence of lysosomal inhibitor indicates an increase of autophagic influx, e. g. more LC3I production and faster conversion to LC3II. Microtubules are polymers of tubulin dimers whose dynamics are regulated by microtubule associated pro teins.