Download scientific diagram | RAPA treatment promoted the expression of ATG7 and E-Cadherin. Immunofluorescent staining against Atg7 and E-Cadherin were performed on the control and RAPA-treated HH4 chick embryos. A-B: The bright-filed images (A) and Atg7 immunofluorescent images (B) of the embryo respectively treated by RAPA at 33 hour. C-C 0 : The transverse sections of Atg7 expression (C) and Atg7 expression C DAPI staining (C 0 ) respectively at the level indicated by dotted line C in B panel, the small panel in C 0 is control section. D-D 0 : The transverse sections of Atg7 expression (D) and Atg7 expression C DAPI staining (D 0 ) respectively at the level indicated by dotted line D in B panel, the small panel in D 0 is control section. E-E 0 : E-Cadherin is expressed on the ectoderm cell membrane of control embryo (white arrow in E 0 ); F-F 0 : E-Cadherin expression level was enhanced on ectoderm cell membrane and ectopic expression in nucleus after RAPA treatment (white arrow in F 0 ). I: Primitive streaks were collected for RT-PCR analysis after being treated with RAPA for 33 h and the control group. In RAPA-treated embryos, E-Cad expression was increased and N-Cad expression was inhibited in comparison with control embryos (N D 20). H: E-Cadherin and N-Cadherin expression levels were detected by realtime PCR. E-Cadherin expression was enhanced and N-Cadherin expression was inhibited in 80 nM RAPA-treated embryos compared to the control. Error bars indicate mean § s.d. ***P < 0.001 indicating highly significant difference between RAPA-treated and control embryos. I: Western blot shows that the expression of E-Cadherin in HCT116 cells from no RAPA treatment (control), or 100 nM RAPA treatment for 1 hour and 2 hours. Actin was used as a loading control. Scale bars D 600 mm in A-B and 500 mm in C-C 0 and 500 mm in D-D 0 , E-E 0 . from publication: Autophagy functions on EMT in gastrulation of avian embryo | Autophagy is important for cell renewing for its contribution to the degradation of bulk cytoplasm, long-lived proteins, and entire organelles and its role in embryonic development is largely unknown. In our study, we investigated the function of autophagy in gastrulation of... | Gastrulation, Chick Embryo and Primitive Streak | ResearchGate, the professional network for scientists.
RAPA treatment promoted the expression of ATG7 and E-Cadherin. Immunofluorescent staining against Atg7 and E-Cadherin were performed on the control and RAPA-treated HH4 chick embryos. A-B: The bright-filed images (A) and Atg7 immunofluorescent images (B) of the embryo respectively treated by RAPA at 33 hour. C-C 0 : The transverse sections of Atg7 expression (C) and Atg7 expression C DAPI staining (C 0 ) respectively at the level indicated by dotted line C in B panel, the small panel in C 0 is control section. D-D 0 : The transverse sections of Atg7 expression (D) and Atg7 expression C DAPI staining (D 0 ) respectively at the level indicated by dotted line D in B panel, the small panel in D 0 is control section. E-E 0 : E-Cadherin is expressed on the ectoderm cell membrane of control embryo (white arrow in E 0 ); F-F 0 : E-Cadherin expression level was enhanced on ectoderm cell membrane and ectopic expression in nucleus after RAPA treatment (white arrow in F 0 ). I: Primitive streaks were collected for RT-PCR analysis after being treated with RAPA for 33 h and the control group. In RAPA-treated embryos, E-Cad expression was increased and N-Cad expression was inhibited in comparison with control embryos (N D 20). H: E-Cadherin and N-Cadherin expression levels were detected by realtime PCR. E-Cadherin expression was enhanced and N-Cadherin expression was inhibited in 80 nM RAPA-treated embryos compared to the control. Error bars indicate mean § s.d. ***P < 0.001 indicating highly significant difference between RAPA-treated and control embryos. I: Western blot shows that the expression of E-Cadherin in HCT116 cells from no RAPA treatment (control), or 100 nM RAPA treatment for 1 hour and 2 hours. Actin was used as a loading control. Scale bars D 600 mm in A-B and 500 mm in C-C 0 and 500 mm in D-D 0 , E-E 0 . Source publicationWenhui LuGuang WangYan Li[...]Xuesong YangAutophagy is important for cell renewing for its contribution to the degradation of bulk cytoplasm, long-lived proteins, and entire organelles and its role in embryonic development is largely unknown. In our study, we investigated the function of autophagy in gastrulation of the chick embryo using both in vivo and in vitro approaches, especially in...Context 1... application of RAPA, the autophagy promoter, we checked Atg7 expression following the treatment of RAPA in early embryo development. At anterior primitive streak, we observed in both epiblast and hypoblast ATG7 were expressed ( Fig. 4C). At the middle primitive streak, in all the ectoderm ATG7 was expressed (Fig. 4D). The ATG7 expression pattern in 3-MA-treated embryos is different from that of the normal Figure 1. Autophagy exists in HH4 chick embryo. A-D: Immunofluorescent staining was performed on whole-mount HH4 chick embryo to detect the expression of Atg7. The ...Context 2... application of RAPA, the autophagy promoter, we checked Atg7 expression following the treatment of RAPA in early embryo development. At anterior primitive streak, we observed in both epiblast and hypoblast ATG7 were expressed ( Fig. 4C). At the middle primitive streak, in all the ectoderm ATG7 was expressed (Fig. 4D). The ATG7 expression pattern in 3-MA-treated embryos is different from that of the normal Figure 1. Autophagy exists in HH4 chick embryo. A-D: Immunofluorescent staining was performed on whole-mount HH4 chick embryo to detect the expression of Atg7. The bright-field images of HH4 chick embryo (A) and the immunofluorescent image of Atg7 ...Context 3... 13 Issue 17 Cell Cycle embryos as we showed in small panels in Fig. 4C 0 and D 0 (Atg7 expressed distinctly in the apical side of epiblast or ectoderm in both levels of ...Context 4... epithelial cell maker, E-Cadherin is distinctly expressed on ectoderm cell membrane in control embryos. However, the E-Cadherin expres- sion lost its normal expression pattern after being treated with 3- MA, and it is slightly enhanced on ectoderm cell membrane after being treated with 3-MA and it shows abnormal expression after RAPA treatment ( Fig. 4F and F 0 ). Furthermore, the extent of E- Cadherin expression was validated by semi-quantitative RT-PCR and quantitative PCR analysis after being treated with RAPA ( Fig. 4G). Meanwhile, we also determined the expression of N-Cadherin, another crucial Cadherin molecule known as meso- derm maker. Using RT-PCR and real-time PCR assays, we dis- ...Context 5... after being treated with 3- MA, and it is slightly enhanced on ectoderm cell membrane after being treated with 3-MA and it shows abnormal expression after RAPA treatment ( Fig. 4F and F 0 ). Furthermore, the extent of E- Cadherin expression was validated by semi-quantitative RT-PCR and quantitative PCR analysis after being treated with RAPA ( Fig. 4G). Meanwhile, we also determined the expression of N-Cadherin, another crucial Cadherin molecule known as meso- derm maker. Using RT-PCR and real-time PCR assays, we dis- covered that N-Cadherin expression was repressed by the treatment of RAPA ( Fig. 4G and H). The real-time PCR results demonstrated that the expression of E-cadherin in ...Context 6... by semi-quantitative RT-PCR and quantitative PCR analysis after being treated with RAPA ( Fig. 4G). Meanwhile, we also determined the expression of N-Cadherin, another crucial Cadherin molecule known as meso- derm maker. Using RT-PCR and real-time PCR assays, we dis- covered that N-Cadherin expression was repressed by the treatment of RAPA ( Fig. 4G and H). The real-time PCR results demonstrated that the expression of E-cadherin in 80 nM RAPA- treated HH4 embryos was 1.3 times compared to the control group. The expression level of N- cadherin in 80 nM RAPA-treated HH4 embryos was 30% lower than the one in the control group. The results of real-time PCR were in accordance with the ...Context 7... PCR were in accordance with the one from RT-PCR assay. To verify our hypothesis in vitro, we treated the HCT116 cells with 100 nM RAPA, which could increase autophagy. E-Cad expression was upregulated after RAPA treatment at 1 hour, compared to that in con- trol. Whereas, E-Cad expression seemed to stay the same at hour 2 after RAPA treatment (Fig. ...Context 8... of E-Cadherin expression in epiblast, even stronger expression in primary streak and in some mesoderm cells. It might be the rea- son for cell accumulations in the bilateral of the primitive groove ( Fig. 5G and H). Meanwhile, we also determined the expression of N-Cadherin by RT-PCR, it was significantly repressed by the treatment of 3-MA (Fig. 4M). Laminin is a major ECM protein component of basal lamina and form sheets in the basal lamina of epithelium as shown in Figure 4I and J. In the embryo exposed to 3-MA, laminin became hardly detectable (Figs. 4K-L), implying the lost of polarity in epiblast layer. Figure 2. Overexpression of Atg7 in epiblast enhanced the expression of ...Context 9... we also determined the expression of N-Cadherin by RT-PCR, it was significantly repressed by the treatment of 3-MA (Fig. 4M). Laminin is a major ECM protein component of basal lamina and form sheets in the basal lamina of epithelium as shown in Figure 4I and J. In the embryo exposed to 3-MA, laminin became hardly detectable (Figs. ...Context 10... 5G and H). Meanwhile, we also determined the expression of N-Cadherin by RT-PCR, it was significantly repressed by the treatment of 3-MA (Fig. 4M). Laminin is a major ECM protein component of basal lamina and form sheets in the basal lamina of epithelium as shown in Figure 4I and J. In the embryo exposed to 3-MA, laminin became hardly detectable (Figs. 4K-L), implying the lost of polarity in epiblast layer. Figure 2. Overexpression of Atg7 in epiblast enhanced the expression of E-Cadherin. Atg7 expression was determined using immunofluorescent staining following the electroporation of Atg7-GFP at half-side of HH4 chick embryo. A: The merge image of Atg7-GFP (green) and E-Cadherin ...Context 11... 13 Issue 17 Cell Cycle embryos to compare Atg7 expression after the inhibition of autophagy, and disclosed that 3-MA treatment led to the disorder of Atg7 expression pattern (Fig. 4A-D and 5A-D), in which Atg7 was also disturbed in all of ectoderm, mesoderm and endo- derm rather than only in apical side of ectoderm and endoderm in normal gastrula embryo (Figs. 1C and D). It indicated that the interruption of Atg7-related autophagy might be involved in the development of 3 germ layers in gastrula embryo, probably through ...Context 12... that autophagy was involved in the EMT progress of some cell lines or tumor tissues, [39][40][41][42] and the embry- onic EMT was similar to the trans- formation of cell phenotype during carcinoma progression. 43 Thus, we did perform the evalua- tion of E-Cadherin, N-Cadherin and laminin expression following the treatment of RAPA and 3-MA (Figs. 4 and 5), and found that exposure to RAPA or 3-MA resulted in disorder of adhesion molecule expression in early chick embryo. One well-known fact is that the E-Cadherin down-regula- tion and N-Cadherin up-regula- tion are necessary conditions for EMT initiation. And the expres- sion of laminin presented on basal side of epithelium should disap- ...Context 13... the expres- sion of laminin presented on basal side of epithelium should disap- pear as well prior to EMT. Thus, we could have reason to assume that our data on adhension mole- cule expression in Figures 4 and 5 is generally in accordance with those conditions indispensable for igniting EMT. In another word, we speculate that autophagy affects the early embryo development principally through regulating EMT, and this also hinted that we could research how the autophagy gene affected tumor invasion by use of chick gastrulation embryo. ...Context 14... could lead to the abnormal expression of E-Cadherin, 49 Therefore, we could speculate that the suppression of Rac1 and Rho (Fig. 6J) is one of the major reasons which lead to the abnormal E-Cadherin expression in 3- MA treated embryos (Fig. 5G) in our study. We could apply same cause to explain the similar phenomena in RAPA treated embryos ( Fig. 4F and F 0 ). Lucy Erin O'Brien et al. reported that dominant-negative Rac1 transfection affected the assemble of Laminin on basolateral membrane, which in turn lead to the invert of polarity in epithelial cells. 50 The reduced-expression of Rac1 in 3-MA treated embryos may contribute to the lost expres- sion of Laminin, which has a crucial effect ...... The EMT is a dynamic process that allows polarised epithelial cells to undergo various biochemical changes and adopt a mesenchymal phenotype [117]. This causes cells to migrate away from the epithelial layer in which they originated and aids in a variety of morphogenetic events during early embryonic development, and also plays a role in wound healing later in life [117,118]. However, in recent decades, this process has been strongly linked to the reprograming of cancer cells towards a more aggressive phenotype with increased stemness. ...Luis Larrea MurilloMegan GreenNiall MahonAlberto SaianiOlga TsigkouCancer initiation and early tumour growth are complex processes influenced by multiple cellular and microenvironmental factors. A critical aspect of tumour progression is the dynamic interplay between cancer cells and the extracellular matrix (ECM), which undergoes significant alterations to support malignancy. The loss of cell polarity is an early hallmark of tumour progression, disrupting normal tissue architecture and fostering cancerous transformation. Circumstantially, cancer-associated microRNAs (miRNAs) regulate key oncogenic processes, including ECM remodelling, epithelial-to-mesenchymal transition (EMT), and tumorigenic vascular development, further driving tumour growth. ECM alterations, particularly changes in stiffness and mechanotransduction signals, create a supportive niche for cancer cells, enhancing their survival, proliferation, and invasion. EMT and its subtype, epithelial-to-endothelial transition (EET), contribute to tumour plasticity, promote the generation of cancer stem cells (CSCs), and support tumour vascularisation. Furthermore, processes of vascular development like vasculogenesis and angiogenesis are critical for sustaining early tumour growth, supplying oxygen and nutrients to hypoxic malignant cells within the evolving cancerous microenvironments. This review explores key mechanisms underlying these changes in tumorigenic microenvironments, with an emphasis on their collective role for tumour initiation and early tumour growth. It will further delve into present in vitro modelling strategies developed to closely mimic early cancer pathophysiology. Understanding these processes is crucial for developing targeted therapies aimed at disrupting key cancer-promoting pathways and improving clinical outcomes.... During EMT in gastrulation, ATG7 expression is observed on the top of the endoderm and ectoderm. E-cadherin, a marker of EMT, increases when ATG7 is overexpressed 194 . ...Jingwei LiuYutong XiaoLiangzi CaoSongming LuLiu CaoAutophagy is a dynamic self-renovation biological process that maintains cell homeostasis and is responsible for the quality control of proteins, organelles, and energy metabolism. The E1-like ubiquitin-activating enzyme autophagy-related gene 7 (ATG7) is a critical factor that initiates classic autophagy reactions by promoting the formation and extension of autophagosome membranes. Recent studies have identified the key functions of ATG7 in regulating the cell cycle, apoptosis, and metabolism associated with the occurrence and development of multiple diseases. This review summarizes how ATG7 is precisely programmed by genetic, transcriptional, and epigenetic modifications in cells and the relationship between ATG7 and aging-related diseases.... Autophagy is involved in cell reprogramming during early development and the transition period from zygote to embryo (Hanna et al., 2010;Chen et al., 2011;Jopling et al., 2011), and is also involved in vertebrate embryonic development (Lu et al., 2014Wang et al., 2015aWang et al., , 2018. Many studies have investigated the role of autophagy in NSCs. ...Yao ShenYi-Piao WangXin ChengXuesong YangGuang WangStem cells are a group of cells with unique self-renewal and differentiation abilities that have great prospects in the repair of spinal cord injury. However, stem cell renewal and differentiation require strict control of protein turnover in the stem cells to achieve cell remodeling. As a highly conserved "gatekeeper" of cell homeostasis, autophagy can regulate cell remodeling by precisely controlling protein turnover in cells. Recently, it has been found that the expression of autophagy markers changes in animal models of spinal cord injury. Therefore, understanding whether autophagy can affect the fate of stem cells and promote the repair of spinal cord injury is of considerable clinical value. This review expounds the importance of autophagy homeostasis control for the repair of spinal cord injury from three aspects-pathophysiology of spinal cord injury, autophagy and stem cell function, and autophagy and stem cell function in spinal cord injury-and proposes the synergistic therapeutic effect of autophagy and stem cells in spinal cord injury.... Therefore, it is important to the autophagy research community that other roles of ATG7, and other key autophagy proteins, are explored further. Alternative functions of ATG7 that have been described include regulating the transcription of cell cycle inhibitor p21 CDKN1A via binding of tumor suppressor p53; promoting E-cadherin expression, resulting in delayed epithelialmesenchymal transition (EMT); and forming a complex with caspase-9, preventing apoptosis and instead leading to necrosis [24][25][26][27][28]. These studies and others have exclusively been performed in mammalian models. ...... Work on chick embryos revealed that ATG7 promotes E-cadherin expression delaying EMT. This was further confirmed in human colorectal carcinoma cells (HCT116) [25]. Interestingly, that study was based on the same ATG7(2) construct as was used in the study on p53 discussed above. ...Valgerdur J. HjaltalinVivian PogenbergKevin OstacoloArnar PálssonMargrét Helga OgmundsdottirThe E1-like enzyme ATG7 belongs to a group of ATG proteins that mediate the autophagy process. Autophagy is a highly conserved degradation pathway important for maintaining homeostasis in eukaryotic cells. Here, we study the evolution of E1 enzymes and specifically describe a region of ATG7 that emerged early in vertebrates. This vertebrate-specific region (VSR) is situated within the adenylation domain of the protein, which is the most conserved domain of E1 enzymes and is of prokaryotic origin. A comparative analysis revealed that ATG7 is unique in this respect, as in other E1 enzyme family members this domain is highly conserved from yeast to humans and has not experienced insertions of extra amino acids. The function of the VSR domain is unknown, but two residues within the region, D522 and S531 have been linked with cancer in humans. Analysis of natural selection indicates positive selection on S531 only on the mammalian clade. Notably, this was the only residue in ATG7 showing this signal. Interestingly, structural analysis of ATG7 predicted that the VSR may be intrinsically disordered and could harbor a macro-molecular binding site. Analysis of cells expressing ATG7 lacking the VSR indicated that these cells are unable to facilitate the lipidation of LC3, suggesting an important role of this region in autophagy. Abbreviations: aBSREL - an adaptive branch-site random effects likelihood; AD - adenylation domain; ATGs - autophagy-related genes; Baf-A1 - Bafilomycin-A1; EV - empty-vector; CTD - C-terminal domain; ECTD - extreme C-terminal domain; EMT - epithelial-mesenchymal transition; FEL - fixed effects likelihood; GABARAP - gamma-aminobutyric acid receptor-associated protein; LC3 - microtubule-associated protein 1A/1B-light chain 3; MEFs - mouse embryonic fibroblasts; MOCS3 - molybdenum cofactor synthesis 3; NTD - N-terminal domain; UBL ubiquitin like protein; VSR - vertebrate specific region... Autophagy plays an important role in the pathogenesis of diverse diseases, including neuronal degeneration, aging and cancer. Inhibition of autophagy resulted in developmental delay via EMT reduction in the gastrulation of chick embryos [21] . Similarly, inhibition of autophagy significantly weakened the TGF-β2 mediated EMT in RPE cells, as reported by Miao et al [22] . ...Yan-Kun YueShan LiuWu LiuAIM: To investigate whether upregulation of apoptosis-stimulating p53 protein 2 (ASPP2) expression could alleviate the development of proliferative vitreoretinopathy (PVR) in a rat model. METHODS: ASPP2-lentivirus or scrambled-lentivirus were transfected into ARPE-19 cells, followed with measurements of cell cytotoxicity by cell counting kit-8 assay. ASPP2 upregulation was confirmed by Western blotting and immunocytochemistry. Then ARPE-19 cells pretreated with ASPP2-lentivirus were intravitreally injected to Brown Norway rats to induce PVR models. PVR development and retinal function were evaluated by retinal photography and electroretinography, respectively. Finally, epithelial-mesenchymal transition as well as autophagy were investigated in rats’ retinas via Western blotting. RESULTS: Protein expression of ASPP2 was significantly upregulated by ASPP2-lentivirus transfection in ARPE-19 cells. The development and progression of PVR were impeded significantly in rats with intravitreal injection of ARPE-19 cells pretreated with ASPP2-lentivirus. Accordingly, retinal functions were less affected and PVR grades were much lower in rats with ASPP2-lentivirus compared to scrambled-lentivirus treatment. Moreover, epithelial-mesenchymal transition and autophagy markers were decreased in the retinas of rats treated with ASPP2-lentivirus. CONCLUSION: ASPP2-lentiviru… truncated (11,116 more characters in archive)