The validation of hPxt1 in vitro function in apoptosis

[Figure 5A] As Pxt1 BH3 was verified to activate the liposome-permeabilizing ability of recombinant Bak, we subsequently investigated the effects of the expression of full-length Pxt1 constructs on the cellular condition of the mitochondria. Therefore, we investigated whether Pxt1 expression causes loss of ??m and release of cytochrome c into the cytosol, the two representative consequences of Bak activation in the mitochondria reflecting the progression of MOMP. First, the ratio of active mitochondria in HeLa cells was measured by flow cytometry using tetramethylrhodamine ethyl ester (TMRE), a cationic dye that accumulates in the mitochondrial matrix and is used as a ??m indicator. [Figure 6D-F and S8] The fate of human or mouse Pxt1-expressing HeLa cells was further analyzed using flow cytometry that determines the rate of apoptosis. [Figure 7A and 8B] We investigated the functional significance of Bak in Pxt1-induced MOMP and apoptosis. To this end, Bax-deficient (Bax-/-) HeLa cell lines were generated using CRISPR. Genetic suppression of Bak expression was achieved by Bak-targeting small interfering RNA (siRNA) called siBak. Bax-/- HeLa cells were transfected with control siRNA (siNON) or siBak for 48 h, and then followed by transfection with the plasmid expressing control GFP or GFP-tagged human Pxt1.
Conclusion:
In this study, we delineated the molecular details and functional consequences of the interaction between Bak and human Pxt1, which was determined to be an effective proapoptotic initiator. Therefore, our work expands our understanding of the regulation of Bak and Bax, the critical executors of MOMP and apoptosis. We speculate that Pxt1 is not unique but is the first case of a noncanonical BH3-only protein activating Bak/Bax. Thus, the identification and molecular characterization of unidentified Bak/Bax-interacting factors would be an interesting topic for future research in this field. We also expect that our research will provide a rational basis for therapeutic approaches, especially those involved in controlling apoptosis that occurs during spermatogenesis.