BikDDA eliminated TNBC cells more powerfully than BikDD.

A. S124A and previous mutant sites T33D and S35D didn’t locate in the pro-apoptotic region of Bik. B. MDA-MB-231 cells were transfected for 28h with the same amount of pUK21, pUK21-BikDD or pUK21-BikDDA. Apoptotic cells were monitored by Annexin V/PI staining and flow-cytometry analysis. The right-lower or right upper quadrant of each plot showed early apoptotic or late apoptotic cells. C. Histogram directly showed the enhanced apoptosis-inducing activity of BikDDA. D. The cytotoxicity of BikDD and BikDDA were analyzed by Cell counting kit and MTT assay (setting at 100% in vector group). E. Western blotting was performed to confirm the prolonged half-life of BikDDA in 231 cells. F. MEK1/2 inhibitor UO126 and selective ERK1/2 inhibitor FR180204 increased the stability of BikDD in 231 cells.