Background Recently, three-dimensional (3D) imaging techniques have been used to detect viral invasion and the appearance of specialized structures established in virus-infected cells. These methods have had a positive impact in the field of virology and helped to further our knowledge of how viruses invade cells. Nearly all positive-strand RNA viruses propagate their viral genomes in part through intracellular membranes. Porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus, accumulates viral RNA that forms replication complexes (RCs) in infected cells. In this study, using immunofluorescence and electron microscopy (EM), we dissected PRRSV-induced membrane structures in infected cells and determined the correlations between PRRSV particles and vesicles stimulated by PRRSV to understand the structural and dynamic aspects of PRRSV infection. Methods We identified the appropriate time point by determining the 50% tissue culture infectious dose (TCID50) and using qRT-PCR and Western blotting. The co-localization of viruses and organelles was determined by immunofluorescence and immune-electron microscopy (IEM). The ultrastructure of cells infected by PRRSV was observed using EM and electron tomography (ET). Results In our study, we found that PRRSV dsRNA was located at the endoplasmic reticulum (ER) and autophagosomes; in addition, the N protein was located at the mitochondria, ER and autophagosomes. Vesicles induced by PRRSV appeared at 16 hours post-infection (h.p.i.) and increased in size with time during the infection period. In addition, our findings demonstrated that the virus vesicles originated from the ER, and these two organelle structures connected with each other to form a reticulovesicular network (RVN) that provided a site for virus replication and assembly. Conclusion Our results revealed that membrane vesicles induced by PRRSV were derived from the ER. The vesicles may provide a location for PRRSV replication and assembly.
Abstract High-mobility group box 2 (HMGB2) is an abundant, chromatin-associated protein that plays an essential role in the regulation of transcription, cell proliferation, differentiation, and tumorigenesis. However, the underlying mechanism of HMGB2 in adipogenesis remains poorly known. Here, we provide evidence that HMGB2 deficiency in preadipocytes impedes adipogenesis, while overexpression of HMGB2 increases the potential for adipogenic differentiation. Besides, depletion of HMGB2 in vivo caused the decrease in body weight, white adipose tissue (WAT) mass, and adipocyte size. Consistently, the stromal vascular fraction (SVF) of adipose tissue derived from hmgb2 −/− mice presented impaired adipogenesis. When hmgb2 −/− mice were fed with high-fat diet (HFD), the body size, and WAT mass were increased, but at a lower rate. Mechanistically, HMGB2 mediates adipogenesis via enhancing expression of C/EBPβ by binding to its promoter at “GGGTCTCAC” specifically during mitotic clonal expansion (MCE) stage, and exogenous expression of C/EBPβ can rescue adipogenic abilities of preadipocytes in response to HMGB2 inhibition. In general, our findings provide a novel mechanism of HMGB2-C/EBPβ axis in adipogenesis and a potential therapeutic target for obesity.
Abstract Single nucleotide polymorphisms (SNPs) widely existing in different breeds genome represent population-specific. Under the influence of long-term evolution and artificial selection, there are a large number of SNPs between western lean-type pig breeds and Chinese indigenous pig breeds, but until now, little is known about their roles in inter-breed differences. Our study revealed SNP rs3471653254 C>T generated from the two types of pigs mentioned above, located in the promoter shared by MyoG and Myoparr, played an important role in the differentiation of myoblast by influencing the enrichment of HOXA5 to regulate the transcription of MyoG and Myoparr. Meanwhile, Myoparr could be used as the sponge of mir-30b-3p which repressed myogenic differentiation and muscle regeneration through targeting MyoD. Our results indicated that SNP rs3471653254 C>T is essential for myogenic differentiation and regeneration and could be used as an ideal site for increasing lean meat production in pigs.
C1q/tumor necrosis factor-related protein 6 (CTRP6), an adipose-tissue secretory factor, plays an important role in inflammatory reaction and carcinogenesis. However, the biological function of CTRP6 in adipogenesis remains unclear. In this study, we examined the effects of CTRP6 knockdown on lipogenesis of 3T3-L1 adipocytes. The results showed that after 3T3-L1 adipocytes transfected with anti-CTRP6 small interfering RNA (siRNA), not only levels of secreted CTRP6 protein in the culture medium but also the expression level of the CTRP6 protein in the 3T3-L1 adipocytes was significantly reduced (P < 0.01). In addition, the number of lipid droplets in the adipocytes was reduced, as well as the OD values reflecting the fat content being significantly decreased (P < 0.01). Meanwhile the levels of adipogenic markers, including peroxisome proliferator activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), CCAAT/enhancer-binding protein β (C/EBPβ) and adipocyte fatty acid-binding protein 4 (aP2), were decreased after treatment with anti-CTRP6 siRNA, whereas the expression of adipose triglyceride lipase (ATGL) and triacylglycerol hydrolase (TGH) were increased. Furthermore, after transfection, activity of phosphorylated Erk1/2 (p-Erk1/2) was inhibited in the early stage of differentiation, but in terminal differentiation of adipocytes, its activity was activated. Taken together, the results indicate that knockdown of CTRP6 can inhibit adipogenesis of 3T3-L1 adipocytes through lipogenic marker genes and Erk1/2 signaling pathway.
Abstract Objectives Mixed lineage leukaemia protein‐1 (MLL1) mediates histone 3 lysine 4 (H3K4) trimethylation (me3) and plays vital roles during early embryonic development and hematopoiesis. In our previous study, we found its expression was positively correlated with embryonic myogenic ability in pigs, indicating its potential roles in mammalian muscle development. The present work aimed to explore the roles and regulation mechanisms of MLL1 in myogenesis. Materials and methods The expression of MLL1 in C2C12 cells was experimentally manipulated using small interfering RNAs (siRNA). 5‐ethynyl‐2′‐deoxyuridine (EdU) assay, cell cycle assay, immunofluorescence, qRT‐PCR and Western blot were performed to assess myoblast proliferation and differentiation. Chromatin immunoprecipitation assay was conducted to detect H3K4me3 enrichment on myogenic factor 5 ( Myf5 ) promoter. A cardiotoxin (CTX)‐mediated muscle regeneration model was used to investigate the effects of MLL1 on myogenesis in vivo. Results MLL1 was highly expressed in proliferating C2C12 cells, and expression decreased after differentiation. Knocking down MLL1 suppressed myoblast proliferation and impaired myoblast differentiation. Furthermore, knockdown of MLL1 resulted in the arrest of cell cycle in G1 phase, with decreased expressions of Myf5 and Cyclin D1. Mechanically, MLL1 transcriptionally regulated Myf5 by mediating H3K4me3 on its promoter. In vivo data implied that MLL1 was required for Pax7‐positive satellite cell proliferation and muscle repair. Conclusion MLL1 facilitates proliferation of myoblasts and Pax7‐positive satellite cells by epigenetically regulating Myf5 via mediating H3K4me3 on its promoter.
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