In addition, TNF increased the binding of GST Zfra with JNK1, NF B and p WOX1, and weakly with p53 in a time related manner. Zfra could not interact with I B , FADD, RIP and Fas. Similarly, in response to UV light, there was an increased binding of GST Zfra with endogenous Zfra, p WOX1 and selleck chemicals Crenolanib JNK1 in SK N SH cells, as determined by pull down analysis. UV light increased the binding of endogenous Zfra with phosphorylated JNK1 in SK N SH cells, as determined by co immunoprecipitation. Transiently overexpressed Zfra sequesters WOX1, NF B, p53 and p ERK in the cytoplasm The above data show that endogenous Zfra may physi cally interacts with endogenous WOX1, NF B and JNK1 2 in non stimulated cells. We examined whether tran siently overexpressed Zfra sequesters these proteins in the cytoplasm and prevents their nuclear translocation by stress stimuli.
SK N SH cells were electroporated with EGFP Zfra or EGFP only, Inhibitors,Modulators,Libraries followed by culturing for 24 hr. Transiently overexpressed EGFP Zfra suppressed nuclear localization of NF B, WOX1, p53, p ERK, but had little or no effect on JNK1 2. TNF induced nuclear translocation of NF B in EGFP control Inhibitors,Modulators,Libraries cells, but this event was blocked in the EGFP Zfra expressing cells. Similarly, in Zfra negative breast MCF7 cells, transiently overexpressed EGFP Zfra suppressed nuclear translocation of NF B and WOX1. Interestingly, transiently overex pressed EGFP Zfra did not have a significant effect on the nuclear localization of WOX1 in Molt4 T cells. However, EGFP Zfra blocked UV light induced WOX1 nuclear translocation in Molt4 T cells.
Zfra counteracts the apoptotic function of WOX1 Next, we examined whether Zfra counteracts the apoptotic function of WOX1. L929 cells were Inhibitors,Modulators,Libraries transfected Inhibitors,Modulators,Libraries with an apoptosis inducing amount of EGFP WOX1, in the pres ence or absence of low doses of EGFP Zfra by CaPO4. In parallel with the above observations, low doses of Zfra enhanced the growth of L929 cells during 24 and 48 hr in culture. Zfra counteracted WOX1 mediated growth suppression and death of L929 cells, as determined using crystal violet staining. Similar results were observed by counting the number of apoptotic nuclei. Protein expression of Inhibitors,Modulators,Libraries EGFP WOX1 and EGFP Zfra were examined by fluorescence microscopy. WOX1 is shown in a perinuclear area, whereas Zfra is expressed in both cytoplasm and nucleus. Similarly, L929 cells were electroporated with WOX1 and or Zfra. These cells were cultured for 24 hr, and then proc essed for DNA fragmentation in agarose gel electrophore sis. WOX1 induced apoptosis was blocked by a non apoptotic table 5 dose of Zfra. Again, L929 cells were then electroporated with apopto sis inducing amounts of WOX1 and or Zfra, followed by culturing for 24 and 48 hr, and then processing for cell cycle analysis by FACS.