Other people have also been not able to detect caveolin two in Caco two cells. So, Caco 2 cells do not have caveolae. We then carried out an experiment to examine the result of MBCD on C. jejuni internalization of Caco 2 cells. Inter estingly, treatment method of Caco two cells with MBCD reduced C. jejuni internalization in the dose dependent method. These effects show that MBCD inhibits C. jejuni internalization irrespective of no matter if the cells possess caveolae structures caveo lin 1. Noteworthy is the fact that MBCD continues to be reported to disrupt all lipid rafts. Transfection of Caco 2 cells having a plasmid that ex presses caveolin 1 cDNA benefits from the right localiza tion of caveolin one and the formation of caveolae. Depending on the sum from the information, the investigators con cluded that caveolin one in Caco two cells behaves very similar to caveolin one expressed in cells that generally express the protein.
To find out selleck should the presence of caveolae may possibly potentiate the quantity of C. jejuni internalized, binding and internalization assays were performed with Caco two cells that have been transfected having a plasmid ex pressing caveolin one. No dif ference was observed in the variety of C. jejuni internalized in Caco 2 expressing caveolin one versus the control cells. Importantly, caveolin one was detected only from the Caco two cells transfected together with the caveolin one containing plasmid, as judged by immunoblot analysis that has a caveolin one specific antibody. An alternative approach to expressing caveolin one in cells that ordinarily never express the protein and have no caveolae would be to use caveolin one knockout cells.
Far more specifically, the 3T3 mouse embryonic fibroblast knock out cell line is homozygous for disruption of the caveolin one gene whereas the 3T3 MEF wild style cell line is Cav 1. We performed C. jejuni binding and internalization with the 3T3 MEF WT and 3T3 selleck Microtubule Inhibitor MEF KO cells. We didn’t observe a big difference from the numbers of C. jejuni bound to or in ternalized from the 3T3 MEF WT versus the 3T3 MEF KO cells. Taken collectively, these benefits are constant together with the proposal that C. jejuni invasion of host cells occurs within a caveolae independent method. MBCD treatment method of HeLa cells disrupts B1 integrin and EGF receptor association To tackle the findings of past reviews, we per formed experiments to determine the mechanistic basis for MBCD inhibition of C. jejuni internalization. We hy pothesized that MBCD prevents the host signaling re sponse triggered by C.
jejuni infection, therefore resulting in a decrease in internalized bacteria, dependant on the fol lowing observations, a remedy of cells with MBCD disrupts all lipid rafts, b a substantial volume of the EGF receptor is localized to lipid rafts, but to not caveolae, c activation of your EGF receptor can be activated from the absence of EGF by its association together with the B1 integrin, and d B1 integrin localization is delicate to cholesterol depletion.