Fig 2: Hypothetical pathway where infectious spleen and kidney necrosis virus (ISKNV)-triggered reactive oxidative species (ROS)/Nrf2 stress signals either modulate the apoptotic Bax/Bak death pathway or reduce viral replication in fish cells. ISKNV shows a unique approach, using the ROS-mediated stress response that can either upregulate the death factor genes Bax and Bak or induce oxidative stress signals in the early viral replication stages in fish cells. In the ISKNV-induced Bax/Bak cell death pathway, these signals are also connected to changes in mitochondrial function, observed as MMP loss (??m) and caspase-9/-3 activation in fish cells. On the other hand, the oxidative stress response can also upregulate the stress transcriptional factor Nrf2 and antioxidant enzymes, such as catalase, Cu/MnSOD, and ZnSOD, which are correlated with the metabolism of ROS, such as superoxide and hydrogen peroxide, and thus the recovery of biological function in proteins or molecules. We found that the ISKNV-induced ROS/Nrf2-mediated Bax/Bak death pathway and viral replication can be inhibited by treatment with antioxidants such as GSH and NAC, providing a new strategy for viral control.

Fig 3: Caspase-9 degrades Bcl-2 and Bcl-xL to enhance Puma-induced apoptosis. (a) HCT116 p53 KO cells were treated with 2 µM UCN-01 at different time points. Treated cells were collected and lysed for detection of Bcl-2 and Bcl-xL degradation. ß-Actin was used as a protein loading Ctrl. (b) Cells were pretreated with zLEHD (20 µM) (a specific caspase-9 inhibitor) for 1 h, and then treated with 2 µM UCN-01 for 48 h. Treated cells were collected and lysed for detection of Bcl-2 and Bcl-xL degradation. ß-Actin was used as a protein loading Ctrl. (c) HCT116 p53 KO cells were stably transfected with HA-Bcl-2 vector and then treated with 2 µM UCN-01 for 48 h. Cells were lysed for detection of Bcl-2 degradation with anti-HA antibody. (d). As described in (c), cells were transfected with HA-Bcl-xL. Cells were lysed for detection of Bcl-xL degradation with anti-HA antibody. ß-Actin was used as a protein loading Ctrl. (e) Cells were treated as described in (c), and then cell apoptosis was detected with Annexin V/PI staining using flow cytometry. (f) As described in (d), cells were stably transfected with HA-Bcl-xL vectors and cell apoptosis was detected with Annexin-positive staining

Fig 4: Alo induces breast cancer cell apoptosis by regulating apoptosis-associated protein expression. Western blotting was performed to analyze the expressions of Bax, Bcl-2, caspase-3 and caspase-9 following Alo (0.1, 0.2 and 0.4 mM) treatment for 24 h. Then the band intensity was quantified by ImageJ software. The results were expressed as the mean ± standard deviation of three independent experiments. *P<0.05 and **P<0.01 vs. non-Alo treated group (0 mM Alo). Alo, Aloperine; Bcl, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Fig 5: Caspase expression. The bar graphs show the relative quantification of caspase-3 (A) and caspase-9 (B) densitometry in the FaDu (left panel) and in the CAL 27 cells (right panel). Each protein is normalized to a-tubulin. Data are expressed as the mean ± SD of three independent experiments. * p = 0.05 vs. control; ° p = 0.05 compared to MMC. *** p < 0.001 vs. control; °°° p = 0.001 compared to MMC. (C) Representative Western blot analysis of caspase-3 and -9 in FaDu (left panel) and CAL 27 cells (right panel). The a-tubulin determination was used as load control. Not-cropped Western blot figures are available in Figure S1.