Fig 1: Autophagy gene and protein expression analysis. (A-C) Box plots of qRT-PCR analysis showing gene expression (Relative to GAPDH) of BECN1, ATG3 and ATG7 in term and preterm placentas. n = 20 term and 20 preterm. Data are presented as median and IQR with minima and maxima. Each dot represents individual subject. (D) STRING protein-protein interaction analysis (STRING, Version 11.5). (E,F) Ratio of gene expression of BECN1 and BAX (E), and BECN1 and BCL2 (F) in term and preterm placentas. n = 20 term and 20 preterm. Data are presented as median and IQR with minima and maxima (G,H). Box plots showing ELISA assay of p62/SQSTM1 (G) and CAPN1 (H) on term and preterm villous placenta tissue lysates. Total protein load at 300 µg/ml for each sample. n = 20 term and 20 preterm. Data are presented as median and IQR with minima and maxima. (I) Spearman Rank test correlation analysis between p62/SQSTM1 and CAPN1 protein concentrations on the entire cohort (n = 40). J. Spearman Rank test correlation analysis between p62/SQSTM1 protein concentrations and baby birth weights on the entire cohort (n = 40). Dotted lines represent 95% CI bands of the best-fit lines (Solid). ns = not significant, Mann-Whitney U test (A–H).
Fig 2: Autophagy process and aging. (a) Plasma p62 and (b) ATG5 levels in in control subjects (CS, n = 150) and atrial fibrillation patients (AF, n = 150) divided into three age groups (<50–60 years; 61–70 years; and >70 years). Data are expressed as median and SD. Intragroup significance: ** p < 0.01; intergroup significance: ## p < 0.01.
Fig 3: A proposed hypothesis of apoptosis and autophagy interactions in preterm birth. The imbalanced BAX/BCL2 expression in the preterm villous placenta increases the release of Caspase 3 and augments intrinsic pathway of apoptosis. The upstream downregulation of IL10 may potentiate downregulation of BCL2 expression leading to BAX/BCL2 imbalance. Caspase 8 is also activated as an indicator of potential extrinsic apoptotic pathway activation. On the other hand, the downregulation of autophagy gene ATG7 results in accumulation of p62 cargo protein within the villous tissue which inhibits the net autophagy flux. The high level of p62 favours accumulation of effector caspases, including Caspase 8 and executes apoptosis. The aggregation of caspases including Caspase 3 degrades essential autophagy-associated proteins, such as Beclin 1, ATG3 and ATG7, which consequently inhibits autophagy. Calpain 1 cleaves ATG5 and Beclin 1 and facilitates accumulation of p62 resulting in net inhibition of autophagy flux. Thus, a vicious cycle may potentially be established between apoptosis and autophagy via the reciprocal stimulatory actions of caspases and p62 within the preterm villous placenta that may have a pathognomonic effect on PTB. The figure is created with BioRender.com.
Fig 4: Serum ATG5 (A) and P62 (B) before (T0) and 2 months (T2M) after mixture intake (n = 10) or no treatment (n = 10) in peripheral artery disease patients. Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01.
Fig 5: Role of Autophagy in TAA(A) Plasma p62 levels in the control group (n = 20) and the thoracic aortic aneurysms (TAA) group (n = 29). Quantification of total soluble and insoluble p62 fractions (B) and relative representative Western blot bands (C) in the control group (n = 3) and the TAA group (n = 7). Expression levels of p62 genes (D) in the control group (n = 2) and the TAA group (n = 7). LC3-II protein expression (E) and relative representative Western blot bands (F) in the control group (n = 20) and the TAA group (n = 29). *P < 0.05; ***P < 0.001.
Supplier Page from Wuhan Fine Biotech Co., Ltd. for Human P62/SQSTM1(Sequestosome-1) ELISA Kit