Evaluating the inhibitory priority of Bcl-xL to Bad, tBid and Bax by using live-cell imaging assay.

Molecular regulatory network among the B cell leukemia-2 (Bcl-2) family proteins is a research hotspot on apoptosis. The inhibitory priority of anti-apoptotic Bcl-2 family proteins (such as Bcl-xL) to pro-apoptotic Bcl-2 family proteins (such as Bad, tBid and Bax) determines the outcome of their interactions. Based on over-expression model system, we here evaluate the inhibitory priority of Bcl-xL to Bad, tBid and Bax by using live-cell imaging assay on cell viability. Fluorescence images of living cells co-expressing CFP-Bcl-xL and YFP-Bad or YFP-tBid or YFP-Bax showed that Bcl-xL markedly inhibited Bad/tBid/Bax-mediated apoptosis, revealing that Bcl-xL inhibits the proapoptotic function of Bad, tBid and Bax. In the case of equimolar co-expression of Bad and CFP-Bcl-xL, the inhibition of Bcl-xL on tBid/Bax mediate-apoptosis was completely relieved. Moreover, co-expression of tBid-P2A-CFP-Bcl-xL significantly relieved the inhibition of Bcl-xL on the pro-apoptotic ability Bax, suggesting that Bcl-xL preferentially inhibits the pro-apoptotic ability of Bad over tBid, subsequently to Bax.