To further verify that paclitaxel could induce autophagy in FLCN deficient cells, we exa mined the p62 expression by Western blot. The reduced p62 level commonly indicates activation of autophagy in cells. During the absence of lysosomal inhibitor bafilomycin A1, we observed that expression of p62 protein was de creased in paclitaxel treated FLCN deficient cells, sugges ting that autophagy was activated along with the p62 protein was degraded by way of autophagy. The p62 level was obviously elevated in FLCN deficient cells taken care of with bafilomycin A1 and paclitaxel, indicating autophagy was blocked by bafilomycin A1 and p62 was accumulated in these cells These results demonstrated that paclitaxel could induce autophagy in FLCN deficient cells. To additional confirm the induction of autophagy in these cells, we examined the autophagosome formation following paclitaxel treatment method applying 3 assays.
To start with, we examined the autophagosome formation with transmission electron microscopy assay. Both pairs of cell lines had been examined soon after paclitaxel remedy. The results showed that in creased autophagosome numbers had been current in FLCN deficient cells. We upcoming examined the formation of autophagosome with the visual appeal with the selleck punctate structures with GFP LC3 assay. We transfected these cells having a GFP LC3 plasmid that ectopically expressed LC3 while in the affected cells. The results showed the FLCN deficient cells exhibited a increased number of punctate structures com pared to FLCN expressing UOK257 two and ACHN sc cells. We even further detected autophagy in cells with monodansyl cadaverine staining assay. Since MDC was demonstrated to have increased affinity for lysosomes, here we utilised it as an auxiliary signifies. Similar to the GFP LC3 assay, we analyzed the formation of autophagosomes below fluorescence microscopy.
Yet again, the FLCN deficient cells displayed significantly increased quantity of punctate structures when compared with corresponding counterparts. These outcomes showed that autophagy was in duced by paclitaxel treatment method in FLCN deficient cells. Paclitaxel induces autophagy in FLCN deficient cells by means of activation of ERK pathway To take a look at the molecular mechanism of paclitaxel in duced selelck kinase inhibitor autophagy in FLCN deficient cells, we examined the alteration from the ERK pathway, and that is recognized to be associated with autophagic regulation in lung can cer cells. As presented in Figure 3, elevated ex pressions of phospho MEK and phospho ERK were detected in FLCN deficient renal cell lines, indicating that absence of FLCN was connected together with the ac tivation of the ERK pathway.