Figure 3. Growth inhibition of endogenous EML4-coiled-coil domain in Ba/F3 cells expressing EML4-ALK. (A) Schematic of EML4-ALK and EML4-coiled-coil (cc) domain interaction analysis using FluoppiTM technology. hAG-EML4-ALK, hAG (homo tetramer Azami-GFP)-tagged EML4-ALK; Ash-EML4-ALK, Ash (homo-oligomerized protein assembly helper)-tagged EML4-ALK; Ash-EML4-cc, Ash-tagged EML4-cc domain. hAG-tagged EML4-ALK (EA) and Ash-tagged EA, or hAG-tagged EA and Ash-tagged cc were co-transfected into Ba/F3 cells (Ba/F3 EA/EA and Ba/F3 EA/cc cells, respectively). Protein-protein interaction between EA-EA or EA-cc can be quantified through fluorescent intensity. In the Ba/F3 EA/EA, tagged EA proteins assemble to make oligomers, and binding of the Ash- and Azami-GFP tags makes large fluorescent foci (Azami-GFP hyper). In the Ba/F3 EA/cc, Azami-GFP tagged EA proteins alone emit fluorescence (homo tetramer Azami-GFP), but only a few large fluorescent foci are formed due to Ash-hAG tag binding (Azami-GFP hyper). (B) Flow cytometric analyses of Ba/F3 EA/EA and Ba/F3 EA/cc. In Ba/F3 EA/EA (left), all cells expressed homo tetramer Azami-GFP (green arrow) and 2.57% of cells expressed Azami-GFP hyper (red arrow). In Ba/F3 EA/cc (right), all cells expressed homo tetramer Azami-GFP (green arrow), but only 0.17% of cells expressed Azami-GFPhyper (red arrow), indicating hyper fluorescent foci formation derived from Azami-GFP-EA and Ash-cc interactions was a rare fraction. X axis; GFP fluorescent intensity. Y axis; PI. (C) Cell growth assay of Ba/F3 EA/EA and Ba/F3 EA/cc. Ba/F3 EA/EA cells that contained oligomerized EA protein served as control. Cell growth of Ba/F3 EA/cc was significantly slower compared with Ba/F3 EA/EA. Error bars: SD. (D) Tumor growth assay in mice models. The animals of both Ba/F3 EA/EA and Ba/F3 EA/cc groups developed tumors. The averaged tumor growth delay of group Ba/F3 EA/cc was higher than that of group EA/EA.
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