Fig 2: Inhibition of Bcl-2 and the Akt signaling pathway were involved in silenced-Livin-induced cell death. (A) STRING analysis revealed Livin and associated protein-protein interactions (confidence mode; http://string-db.org/). Within this cluster, Bcl-2 and Akt, which were located in the key nodes and mutually interacted, were selected for further analysis. (B) HCT116 and SW620 cells were incubated with 20 µM 5-FU for 24 h, harvested and subjected to western blot analysis to detect the protein expression level of Bcl-2, p-Akt and T-Akt from the control, NC and shLivin groups. Images are representative of three independent experiments. Histograms represent p-Akt, T-Akt and Bcl-2 protein expression levels quantified by western blotting and the optical analysis software ImageJ in (C) HCT116 and (D) SW620 cells (*P<0.05, shLivin group vs. control or NC groups). Data are presented as the mean ± standard deviation of three independent experiments. Bcl-2, B-cell lymphoma-2; LC3, light chain 3; SMAC, Schizont membrane-associated cytoadherence protein; Akt1, protein kinase B 1; p, phosphorylated; T, total; NC, negative control; shLivin, lentivirus-short hairpin Livin; STRING, Search Tool for the Retrieval of Interacting Genes/Proteins; 5-FU, 5-fluorouracil.

Fig 3: MiR‐130 inhibits hypoxia‐induced PASMC apoptosis by regulating PPARγ expression. (A–C) Western blotting for Bax, Bcl‐2, cleaved caspase‐3, caspase‐3 and apoptosis‐inducing factor (AIF) expression in PASMCs in Nor, Hyp, Hyp+miR‐130 inhibitor and Hyp+miR‐130 inhibitor+siPPARγ groups, β‐actin was used as a loading control (n = 4). (D‐F) The expression levels of cytochrome c (Cyt C) in mitochondrial and cytosol pellets in PASMCs were examined by western blotting with antibodies against Cyt C with COX IV as a mitochondria marker and β‐actin as the internal control (n = 4). (G) The apoptosis index of PASMCs in each group was measured by TUNEL assay (n = 10) (×200; scale bars indicate 100 µm) and is shown as the ratio of TUNEL positive cells (red) to total cells (blue). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01

Fig 4: Representative photomicrographs of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and semiquantitative analysis (A). Immunoblotting and quantitative analyses of Bcl-2, Bax, and cleaved caspase-3 (B). Bcl-2, B-cell lymphoma-2; Bax, Bcl2-associated X; UUO, unilateral ureteral obstruction; LC, L-carnitine. ap < 0.01 vs. sham, bp < 0.05 vs. UUO7, cp < 0.05 vs. UUO14.

Fig 5: Schematic of the interrelationships among p-AMPK, p-mTOR, p-ULK1, autophagy and apoptosis. Accumulated ROS results in the phosphorylation and activation of AMPK. Activated AMPK suppresses mTOR, thereby activating autophagy and exerting an anti-apoptotic function. Additionally, AMPK may directly activate autophagy through other mechanisms. However, excessive ROS and metabolites lead to impairment of AMPK activity, and increased activity of mTOR phosphorylates ULK1 at Ser 757, leading to the suppression of autophagy initiation. Autophagy and BCL2 cooperate against apoptosis and delay the process of senescence. AMPK, 5′ AMP-activated protein kinase; mTOR, mechanistic target of rapamycin; p, phosphorylated; ROS, reactive oxygen species; BCL2, B-cell lymphoma 2; NS, normal saline; D-gal, D-galactose.