Treatment with 5 mM SB202190-induced vacuolation reproducible  in various cell lines, w While it is not. In other cells Induction of vacuoles , since they also detected in non-transformed cell lines and primary Re cells and could not be detected in some of the tested transformed cells. Vacuoles were clearly visible in the sensitive cell lines after 2 h SB202190 treatment. SB202190 induced Anh Ufung of enlarged Erten autophagolysosomes in HT29 cells Previous studies of the induction of cancer c Lon specific autophagic vacuoles and cell death were reported by SB202190. Since this effect was analyzed and characterized Autophagic for its main features in detail in HT29 human cancer cell c Lon, we set the effect of SB202190 in this cell line to be analyzed.
Autophagy is characterized by cytoplasmic material trapping membrane autophagosomes Se and degradation by lysosomal pathway. The ultrastructures autophagosomes are usually often visualized by electron microscopy and therefore purchase huge vacuoles induced by SB202190 probably artifacts of immature autophagic response. As acridine orange and neutral ROTF Staining showed SB202190 treated HT29 cells, vacuoles were very sour. SB202190 induced vacuoles were also positive for the presence of the lysosomal membrane protein LAMP2. The induction of autophagy is cha through the conversion of microtubule protein in Only slightly to form a 3 lipid conjugated LC3 II autophagic vacuoles and recruitment form a pattern distinct F Punctate staining used. After SB202190 treatment, YFP transfected LC3 showed marked point–Shaped structures in the north See the enlarged vacuoles Ert, but not together locate with vacuoles.
This is comparable to the abnormal autophagolysosomes in Sertoli cells w During treatment with lindane observed. since these vacuoles positive LAMP2, S lysosomal acid in origin and therefore we are the reaction induced vacuolation SB202190 assessment of vacuole S uregehalt quantified by acridine orange. For this reason, we treated HT29 cells stained SB202190 and measured red fluorescence of AO by FACS. A significant upregulation zeitabh-Dependent red fluorescence of the AO in response to SB202190 treatment could be clearly demonstrated. We also examined endogenous LC3 in HT29 cells by Western blot. There is a high degree to LC3 II base in HT29 cells that have not increased 2 h SB202190 treatment ht.
However, the treatment leads to lysosomal protease inhibitor SB202190 further accumulation of lipid conjugate LC3 what. To the presence of an active autophagic flux Other markers of autophagy response as Beclin 1 and ATG5 ATG12 showed no significant Ver Change. Interestingly, treatment of HT29 cells with rapamycin, an mTOR inhibitor, and an inducer of autophagy commonly used, also showed Similar autophagic turnover, such as by LC3 II / I ratio LC3 ratio measured in the presence and absence of the protease inhibitor. It is interesting to note that rapamycin did not induce the formation of vacuoles. In agreement with previous studies, treatment with 3 methyl groups, k Nnte adenine, an inhibitor of PI3-kinase class III and induces autophagy inhibitor SB202190 to suppress vacuolation.