Next Ally, chloroquine, an inhibitor of lysosomal Ans Acidification and wortmannin, an inhibitor of PI3 kinase broad spectrum, prevented the formation of vacuoles. Sun appear PI3 NVP-LDE225 LDE225 kinase, LC3 conjugation of lipids and acidification of lysosomes before the vacuolization response. SB202190 treatmen and accumulation of autophagy substrates involvement of p38 in autophagy regulation of complex and various studies have suggested r Gegens tze In the way of starvation-induced autophagy. Therefore, it was assumed that the differences between rapamycin and SB202190 Nnte k Due to zinc Siege effect of inhibition of p38 that Bl Cke autophagolysosome Aufl acids Solution, and thus the accumulation of vacuoles S.
To prove this hypothesis, we monitored the levels of p62, a protein, substrate, preferably degraded by autophagy in SB202190 and rapamycin-treated cells in parallel. Rapamycin no significant effect on endogenous p62 protein levels in HT29 cells, w During SB202190 strongly too late to p62 levels in HT29 cells Times lower regulated. LC3 conjugated lipid accumulated after 12 and 24 h treatment SB202190 carrying a model of conjugation and increased Hte lipid Stromreduzierungsger t of lysosomal degradation. We have also verified by analysis of the turnover of autophagic LC3 II and p62 in the presence or absence of leupeptin, a lysosomal protease inhibitor. When HT29 cells were treated with SB202190, an accumulation of p62 and zeitabh LC3 II-dependent was independently Ngig observed by the presence or absence of leupeptin.
In contrast, rapamycin treatment alone showed no significant effects on the levels of LC3 II or p62. Rapamycin, in combination with leupeptin LC3 strong combination of lipids and regulations small but significant p62. This observation shows fa Conclusively that in the same cell system, the Ums Autophagy substrates rapamycin affected differently and SB202190 tze. Rapamycin induces autophagy proteolysis sensitive active treatment leupeptin, w During SB202190 induces autophagy non-productive accumulation of p62 and LC3 II was Zus Tzlich N Hrstoffmangel induced autophagic proteolysis also significantly reduced by SB202190 treatment. SB202190 does not induce de novo expression of the gene in vacuole formation HT29 ben SB202190 reaction induced autophagic vacuoles and accumulation CONFIRMS was previously reported that autophagic transcriptional reprogramming leading to gene regulation Pro, r connected which is not at this time been established.
Therefore, we decided to characterize the influence of insurance Changes in gene expression in SB202190-induced vacuole formation. 24 hours SB202190 treatment resulted Erh Increase of small, but consistent in expressing GABARAP, a homologue of LC3 and BNIP3L, a protein in autophagy and apoptosis are involved. This up-regulation of gene transcription and GABARAP been reported there this to the activation FOXO3a. To monitor the Transkriptionsaktivit t of Foxo, we generated stable, Chape response element integrated reporter HT29 cells. Interestingly, SB202190 induced reporter activity T FHREluc only observed after 48 h SB202190 treatment.