Fig 2: B2 protein induces ROS/Nrf2-mediated redox oxidative stress signals via stress markers catalase, Cu/ZnSOD, and MnSOD in NOD/SCID mice. RT-qPCR analysis of oxidative stress signal via stress markers (A) Nrf2, (B) catalase, (C) Cu/ZnSOD, and (D) MnSOD, suggesting the induction of a ROS/Nrf2-mediated redox oxidative stress signal in solid tumors (N = 4–6). The data are representative of three separate experiments, and the error bars represent the SEMs. * p < 0.05 and ** p < 0.01, as analyzed by one-way ANOVA with Tukey’s multiple comparisons. (E) Upregulation of B2-mitochondria-targeting-induced catalase, MnSOD, and Cu/ZnSOD expression in A549 cell solid tumors according to Western blot analysis. (F–I) Quantification of the results in E. The data are representative of three separate experiments, and the bars represent the SEMs. * p < 0.05 and ** p < 0.01, NS = not significant, as analyzed by one-way ANOVA with Tukey’s multiple comparisons.

Fig 3: Hypothesized effect of mitochondrion-targeting protein B2 on multiple signaling pathways by ROS-mediated oxidative stress in cancer cells. The expression of the B2 gene in A549 lung tumor cells or in A549 cell-induced solid tumors in NOD/SCID mice induced ROS-mediated stress signals, causing p53/Bax-mediated apoptosis and RIPK3-mediated necroptosis. The B2 protein can transfer into the mitochondria using its novel transferring signal sequence. 41RTFVISAHAA50. It then regulates complex II activity, suppressing ATP production and enhancing ROS generation, thereby repressing cancer cell growth. B2 triggers ROS/Nrf2-mediated stress, further affecting multiple signaling pathways, such as (1) the induction of the p53- and Bax-mediated apoptotic signals and the RIPK3-mediated necroptosis signal, as well as (2) a reduction in the cancer marker expressions of CD133, Thy1, and napsin in mice, which are responsible for cell migration and invasion in lung tumors.

Fig 4: B2 protein causes an ROS/Nrf2-mediated redox oxidative stress signal following mitochondrial targeting in A549 cells. (A) Schematic representation of B2-induced, ROS/Nrf2-mediated stress response in A549 cells 48 h post transfection. (B) Identification of B2-induced ROS production in different A549 cells transfected with Flag, Flag-B2, and Flag-ΔB2 plasmids. ROS production was traced using the DCFDA method. (C) Quantification of ROS production. The data are representative of three separate experiments, and the bars represent the SEMs. * p < 0.05 and ** p < 0.01, as analyzed by one-way ANOVA with Tukey’s multiple comparisons. (D) Identification of p38 phosphorylation sites and Nrf2 expression in A549 cells by Western blot analysis. (E,F) Quantification of the results in D. The data are representative of three separate experiments, and the error bars represent the SEMs. * p < 0.05 and ** p < 0.01, as analyzed by one-way ANOVA with Tukey’s multiple comparisons.