scholarly journals Beclin 1, LC3 and P62 Expression in Equine Sarcoids

Animals ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 20
Author(s):  
Manuela Martano ◽  
Gennaro Altamura ◽  
Karen Power ◽  
Pierluigi Liguori ◽  
Brunella Restucci ◽  
...  
Keyword(s):  
Western Blot ◽  
Normal Skin ◽  
Beclin 1 ◽  
Autophagic Flux ◽  
Damaged Material ◽  
Complex Process ◽  
Causative Agents ◽  
No Activation ◽  
Skin Samples ◽  
Selection Of

Background: It is well known that δ-bovine papillomaviruses (BPV-1, BPV-2 and BPV-13) are one of the major causative agents of equine sarcoids, the most common equine skin tumors. Different viruses, including papillomaviruses, evolved ingenious strategies to modulate autophagy, a complex process involved in degradation and recycling of old and damaged material. Methods: The aim of this study was to evaluate, by immunohistochemistry (IHC) and Western blot (WB) analysis, the expression of the main related autophagy proteins (Beclin 1, protein light chain 3 (LC3) and P62), in 35 BPV1/2 positive equine sarcoids and 5 BPV negative normal skin samples. Results: Sarcoid samples showed from strong-to-moderate cytoplasmic immunostaining, respectively, for Beclin 1 and P62 in >60% of neoplastic fibroblasts, while LC3 immunostaining was weak to moderate in ≤60% of neoplastic fibroblasts. Western blot analysis confirmed the specificity of the antibodies and revealed no activation of autophagic flux despite Beclin 1 overexpression in sarcoid samples. Conclusion: Results could suggest the activation of the initial phase of autophagy in equine sarcoids, and its impairment during the following steps. The impairment of autophagy could lead to a selection of a quiescent population of fibroblasts, which survive longer in a hypoxic microenvironment and produced more and/or altered collagen.

scholarly journals A two-step model of autophagy: autophagosome formation, degradation and net turnover

2020 ◽  
Author(s):  
Ainhoa Plaza-Zabala ◽  
Virginia Sierra-Torre ◽  
Amanda Sierra
Keyword(s):  
Western Blot ◽  
Autophagic Flux ◽  
Autophagosome Formation ◽  
Autophagy Flux ◽  
Gold Standard Method ◽  
Complex Process ◽  
Step Model ◽  
Using Data ◽  
Definition Of ◽  
The Brain

AbstractAutophagy is a complex process that encompasses the enclosure of cytoplasmic debris or dysfunctional organelles in membranous vesicles, the autophagosomes, for their elimination in the lysosomes. A gold-standard method to assess its induction is the analysis of the autophagic flux using as a surrogate the expression of the microtubule-associated light chain protein 3 conjugated to phosphatidylethanolamine (LC3-II) by Western blot, in the presence of lysosomal inhibitors. Therefore, the current definition of autophagy flux actually puts the focus on the degradation stage of autophagy. In contrast, the most important autophagy controlling genes that have been identified in the last few years in fact target early stages of autophagosome formation. From a biological standpoint is therefore conceivable that autophagosome formation and degradation are independently regulated and we argue that both stages need to be systematically analyzed. Here, we propose a simple two-step model to understand changes in autophagosome formation and degradation using data from conventional LC3-II Western blot, and test it using two models of autophagy modulation in cultured microglia, the brain macrophages: rapamycin and the ULK1/2 inhibitor, MRT68921. Our model provides a comprehensive understanding of the autophagy process and will help to unravel the effect of genetic, pharmacological, and environmental manipulations on both the formation and degradation of autophagosomes.

scholarly journals Assessing Autophagy in Microglia: A Two-Step Model to Determine Autophagosome Formation, Degradation, and Net Turnover

2021 ◽  
Vol 11 ◽  
Author(s):  
Ainhoa Plaza-Zabala ◽  
Virginia Sierra-Torre ◽  
Amanda Sierra
Keyword(s):  
Western Blot ◽  
Cell Types ◽  
Autophagic Flux ◽  
Immune Functions ◽  
Autophagosome Formation ◽  
Gold Standard Method ◽  
Complex Process ◽  
Step Model ◽  
Using Data ◽  
Definition Of

Autophagy is a complex process that encompasses the enclosure of cytoplasmic debris or dysfunctional organelles in membranous vesicles, the autophagosomes, for their elimination in the lysosomes. Autophagy is increasingly recognized as a critical process in macrophages, including microglia, as it finely regulates innate immune functions such as inflammation. A gold-standard method to assess its induction is the analysis of the autophagic flux using as a surrogate the expression of the microtubule-associated light chain protein 3 conjugated to phosphatidylethanolamine (LC3-II) by Western blot, in the presence of lysosomal inhibitors. Therefore, the current definition of autophagy flux actually puts the focus on the degradation stage of autophagy. In contrast, the most important autophagy controlling genes that have been identified in the last few years in fact target early stages of autophagosome formation. From a biological standpoint is therefore conceivable that autophagosome formation and degradation are independently regulated and we argue that both stages need to be systematically analyzed. Here, we propose a simple two-step model to understand changes in autophagosome formation and degradation using data from conventional LC3-II Western blot, and test it using two models of autophagy modulation in cultured microglia: rapamycin and the ULK1/2 inhibitor, MRT68921. Our two-step model will help to unravel the effect of genetic, pharmacological, and environmental manipulations on both formation and degradation of autophagosomes, contributing to dissect out the role of autophagy in physiology and pathology in microglia as well as other cell types.

An Insight into the Role of Apoptosis and Autophagy in Nitric Oxide–Induced Articular Chondrocyte Cell Death

Cartilage ◽  
2020 ◽  
pp. 194760352097676
Author(s):  
Ekkapol Akaraphutiporn ◽  
Takafumi Sunaga ◽  
Eugene C. Bwalya ◽  
Wang Yanlin ◽  
Mwale Carol ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Death ◽  
Western Blot ◽  
Cell Apoptosis ◽  
Molecular Mechanisms ◽  
Interleukin 1 ◽  
Chondrocyte Apoptosis ◽  
Protective Mechanism ◽  
Autophagic Flux ◽  
Qpcr Analysis

Objective To investigate the role and characterize the molecular mechanisms regulating apoptosis and autophagy in nitric oxide (NO)–induced chondrocyte cell death. Design Cell apoptosis and autophagy were evaluated in chondrocytes treated with sodium nitroprusside (SNP) combined with the presence or absence of interleukin-1 beta (IL-1β) and nutrient-deprived conditions. The concentration of nitrite was determined by Griess reaction. Activation of apoptosis and autophagy were determined by immunocytochemistry, Western blot, and quantitative real-time polymerase chain reaction (qPCR) analysis. Flow cytometry and MTT assay were used to assess cell viability. Results Cotreatment of chondrocytes with SNP and IL-1β under nutrient-deprived condition potentially enhanced the effect of NO-induced cell death. Immunocytochemistry, Western blot, and qPCR analysis indicated that treatment of chondrocytes with SNP significantly reduced autophagic activity, autophagic flux, and multiple autophagy-related (Atg) genes expression. These findings were associated with an increase in ERK, Akt, and mTOR phosphorylation, whereas autophagy induction through mTOR/p70S6K inhibition by rapamycin significantly suppressed NO-induced cell apoptosis. Furthermore, the cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-3 activation in response to apoptosis was weakly detected. These results corresponded with a significant increase in apoptosis-inducing factor (AIF) expression, suggesting the involvement of the caspase-independent pathway. Conclusions These results demonstrate that in chondrocyte cultures with cells induced into an osteoarthritis state, NO inhibits autophagy and induces chondrocyte apoptosis mainly, but not completely through the caspase-independent pathway. Our data suggest that autophagy is a protective mechanism in the pathogenesis of osteoarthritis and could be proposed as a therapeutic target for degenerative joint diseases.

scholarly journals Selective STAT3 Inhibitor Alantolactone Ameliorates Osteoarthritis via Regulating Chondrocyte Autophagy and Cartilage Homeostasis

2021 ◽  
Vol 12 ◽  
Author(s):  
Wenbin Pei ◽  
Xiaojian Huang ◽  
Bowei Ni ◽  
Rui Zhang ◽  
Guangyi Niu ◽  
...  
Keyword(s):  
Western Blot ◽  
Inflammatory Responses ◽  
Cartilage Degeneration ◽  
Cartilage Destruction ◽  
Signaling Molecules ◽  
Inflammatory Factors ◽  
Signal Pathways ◽  
Autophagic Flux ◽  
Safranin O

Osteoarthritis (OA), which is identified by chronic pain, impacts the quality of life. Cartilage degradation and inflammation are the most relevant aspects involved in its development. Signal transducer and activator of transcription 3(STAT3), a member of the STATs protein family, is associated with inflammation. Alantolactone (ALT), a sesquiterpene lactone compound, can selectively suppress the phosphorylation of STAT3. However, the pharmacological effect of ALT on OA is still imprecise. In this study, IL-1β (10 ng/ml) was applied to cartilage chondrocytes, which were treated with different concentrations of Alantolactone for 24 h. The expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2(COX2), matrix metalloproteinases (MMPs) and thrombospondin motifs-5 (ADAMTS5) were detected by western blot. Protein expression of Collagen Ⅱ was observed by western blot, safranin O staining and immunofluorescence. Manifestation of autophagy related proteins such as autophagy-related gene-5 (ATG5), P62, LC3Ⅱ/Ⅰ and PI3K/AKT/mTOR-related signaling molecules were measured by western blot and autophagic flux monitored by confocal microscopy. Expression of STAT3 and NF-κB-related signaling molecules were evaluated by western blot and immunofluorescence. In vivo, 2 mg/kg ALT or equal bulk of vehicle was engaged in the destabilization of medial meniscus (DMM) mouse models by intra-articular injection, the degree of cartilage destruction was classified by Safranin O/Fast green staining. Our findings reported that the enhance of inflammatory factors containing iNOS, COX2, MMPs and ADAMTS5 induced by IL-1β could be ameliorated by ALT. Additionally, the diminish of Collagen Ⅱ and autophagy which was stimulated by IL-1β could be alleviated by ALT. Mechanistically, STAT3, NF-κB and PI3K/AKT/mTOR signal pathways might be involved in the effect of ALT on IL-1β-induced mouse chondrocytes. In vivo, ALT protected cartilage in the DMM mouse model. Overall, this study illustrated that ALT attenuated IL-1β-induced inflammatory responses, relieved cartilage degeneration and promoted impaired autophagy via restraining of STAT3 and NF-κB signal pathways, implying its auspicious therapeutical effect for OA.

scholarly journals 9-PAHSA Improves Cardiovascular Complications by Promoting Autophagic Flux and Reducing Myocardial Hypertrophy in Db/Db Mice

2021 ◽  
Vol 12 ◽  
Author(s):  
Yan-Mei Wang ◽  
Shou-Ling Mi ◽  
Hong Jin ◽  
Qi-Lin Guo ◽  
Zhong-Yu Yu ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Blood Glucose ◽  
Western Blot ◽  
Vascular Calcification ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Myocardial Hypertrophy ◽  
Autophagic Flux ◽  
Glucose Levels ◽  
Blood Glucose Levels

Atherosclerotic cardiovascular disease is a common and severe complication of diabetes. There is a large need to identify the effective and safety strategies on diabetic cardiovascular disease (DCVD). 9-PAHSA is a novel endogenous fatty acid, and has been reported to reduce blood glucose levels and attenuate inflammation. We aim to evaluate the effects of 9-PAHSA on DCVD and investigate the possible mechanisms underlying it. Firstly, serum 9-PAHSA levels in human were detected by HPLC-MS/MS analysis. Then 9-PAHSA was synthesized and purified. The synthesized 9-PAHSA was gavaged to db/db mice with 50 mg/kg for 4 weeks. The carotid arterial plaque and cardiac structure was assessed by ultrasound. Cardiac autophagy was tested by western blot analysis, electron microscope and iTRAQ. The results showed that 9-PAHSA, in patients with type 2 diabetes mellitus (T2DM), was significantly lower than that in non-diabetic subjects. Administration of 9-PAHSA for 2 weeks reduced blood glucose levels. Ultrasound observed that continue administration of 9-PAHSA for 4 weeks ameliorated carotid vascular calcification, and attenuated myocardial hypertrophy and dysfunction in db/db mice. Electron microscopy showed continue 9-PAHSA treatment significantly increased autolysosomes, while dramatically decreased greases in the myocardial cells of the db/db mice. Moreover, iTRAQ analysis exhibited that continue 9-PAHSA treatment upregulated BAG3 and HSPB8. Furthermore, western blot analysis confirmed that 9-PAHSA down-regulated Akt/mTOR and activated PI3KIII/BECN1 complex in diabetic myocardium. Thus, 9-PAHSA benefits DCVD in diabetic mice by ameliorating carotid vascular calcification, promoting autophagic flux and reducing myocardial hypertrophy.

scholarly journals Defective Autophagy in MPN Were Predominantly Detected in Megakaryocytes in Philadelphia Negative (Ph -) Myeloproliferative Neoplasm

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5386-5386
Author(s):  
Jen-Chin Wang ◽  
Guanfang Shi ◽  
Karan Josan ◽  
Preethi Ramachandran ◽  
Vladimir Gotlieb ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Western Blot ◽  
Cell Signaling ◽  
Mononuclear Cells ◽  
Myeloproliferative Neoplasm ◽  
Beclin 1 ◽  
Positive Staining ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear ◽  
Cytoplasmic Staining

We previously reported that autophagy was defective in classical Philadelphia negative (Ph -) Myeloproliferative Neoplasm (MPN) by demonstrating increased p62, decreased Beclin-1 by RT-PCR and Western Blot (WB) methods and decreased LC3-II by WB (ASH annual meeting poster 2018) in peripheral blood mononuclear cells. Now, we further performed immunohistochemical staining of these autophagy markers on the bone marrow biopsy specimens. Methods: Formalin-fixed and paraffin-embedded bone marrow samples were stained with primary antibodies against Beclin-1 (mouse monoclonal, Millipore), LC3B (rabbit monoclonal, Cell Signaling Technology), and p62 (mouse monoclonal, Cell Signaling Technology). The tissue sections were analyzed using VENTANA BenchMark Ultra System (Ventana Medical Systems, Inc.) according to the manufacturer's protocol. Patients who were studied included 12 essential thrombocythemia (ET), 10 polycythemia (PV), and 5 myelofibrosis (MF) (including 1 post-ET MF and 1 post-PV MF). Scoring was given based on the degree of cytoplasmic staining. Score 1 included weak cytoplasmic stain, score 2 included moderate cytoplasmic stain and score 3 was given for strong cytoplasmic staining . Results: 1) As shown in Fig1, the predominant cells which stained positive including p62, Beclin-1 , or LC3 B were mostly found on the megakaryoctes, although very few of other cell types were found to have positive staining as well 2) As shown in Table 1, the immunostaining results were in general correlated to the RT-PCR , or western blot findings that a defective autophagy was demonstrated in MPN patients with combined finding of a stronger staining in p62,and weak staining in Beclin-1 and LC3B. Conclusion: We have demonstrated defective autophagy in these Ph (-) MPN, by immunostaining of the bone marrow specimens, and demonstrating positivity for defective autophagy predominantly in megakaryocytes. Since megakaryocytes are the most important cells involved in the pathogenesis of MPN, we propose that defective autophagy in megakaryocytes play an important role in the pathogenesis of these diseases. Disclosures Wang: Incyt: Research Funding.

Knockdown of BAG3 synergizes with olaparib to kill ovarian cancer cells via repressing autophagy

2020 ◽  
pp. jim-2020-001602
Author(s):  
Kexin Wang ◽  
Jianhua Zheng
Keyword(s):  
Ovarian Cancer ◽  
Western Blot ◽  
Transfection Efficiency ◽  
Parp Inhibitor ◽  
Beclin 1 ◽  
Cell Counting ◽  
Ovarian Cancer Cells ◽  
Western Blot Assay ◽  
Related Proteins

This study aimed at expounding the synergistic effect of Bcl-2-associated athanogene 3 (BAG3) knockdown and poly ADP-ribose polymerase (PARP) inhibitor on ovarian cancer (OC) cells and the potential mechanism. Short hairpin RNA (shRNA) targeting BAG3 (sh-BAG3) was transfected into SK-OV-3 (SKOV-3 ;SKOV3) and A2780 cells, and western blot assay was used to detect transfection efficiency. Cell proliferation and apoptosis were detected by the cell counting kit-8 method, 5-Bromodeoxyuridine (BrdU) experiment and flow cytometry analysis, respectively. The expressions of apoptosis-related proteins Bax and Bcl-2, as well as the expressions of autophagy-related proteins LC3-I, LC3-II and Beclin-1, were examined by western blot assay. Additionally, the cells were treated with autophagy activator rapamycin to investigate whether the tumor-suppressive function of BAG3 knockdown+PARP inhibitor was dependent on autophagy. In this work, we demonstrated that BAG3 knockdown further sensitized OC cells to olaparib treatment, reducing cellular viability and promoting apoptosis. Both sh-BAG3 and olaparib decreased the expression of Beclin-1 and the LC3-Ⅱ:LC3-I ratio, and their synergism further inhibited the process of autophagy. However, the aforementionede effects were reversed after the cells were treated with rapamycin. Based on these results, we concluded that BAG3 knockdown synergizes with olaparib to kill OC cells in vitro by repressing autophagy.

scholarly journals Moving from Evaluation to Trial: How do SMEs Start Adopting Cloud ERP?

2015 ◽  
Vol 19 ◽  
Author(s):  
Siti Aisyah Salim ◽  
Darshana Sedera ◽  
Sukanlaya Sawang ◽  
Abdulrahman Hamad E Alarifi ◽  
Maura Atapattu
Keyword(s):  
Theory Of Planned Behaviour ◽  
Selection Process ◽  
Planned Behaviour ◽  
Intention To Adopt ◽  
Multi Stage ◽  
Complex Process ◽  
Stage Process ◽  
It Applications ◽  
Multiple Stages ◽  
Selection Of

The advent of cloud technology involving low subscription overheads cost has provided small and medium-sized enterprises (SMEs) with the opportunity to adopt new cloud-based corporate-wide systems (i.e., cloud ERP). This technology, operating through subscription-based services, has now provided SMEs with a complete range of IT applications that were once restricted to larger organisations. As anecdotal evidences suggest, SMEs are increasingly adopting cloud-based ERP software. The selection of an ERP is a complex process involving multiple stages and stakeholders, suggesting the importance of closer examination of cloud ERP adoption in SMEs. Yet, prior studies have predominantly treated technology adoption as a single activity and largely ignored the issue of ERP adoption in SMEs. Understanding of the process nature of the adoption and the factors that are important in each stage of the adoption potentially may result in guiding SMEs to make well-informed decisions throughout the ERP selection process. Thus, our study proposes that the adoption of cloud ERP should be examined as a multi-stage process. Using the Theory of Planned Behaviour (TPB) and Ettlie’s adoption stages, as well as employing data gathered from 162 owners of SMEs, our findings show that the factors that influence the intention to adopt cloud ERP vary significantly across adoptive stages.

scholarly journals Attenuating Hypoxia-Induced Apoptosis and Autophagy of Mesenchymal Stem Cells: the Potential of Sitagliptin in Stem Cell-Based Therapy

2015 ◽  
Vol 37 (5) ◽  
pp. 1914-1926 ◽  
Author(s):  
Xi-Mei Wang ◽  
Yue-Jin Yang ◽  
Yong-Jian Wu ◽  
Qian Zhang ◽  
Hai-Yan Qian
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Western Blot ◽  
Annexin V ◽  
Beclin 1 ◽  
Dipeptidyl Peptidase 4 ◽  
Acridine Orange Staining ◽  
Cell Based Therapy ◽  
Induced Apoptosis ◽  
The Impact

Background/Aims: Dipeptidyl peptidase-4 (DPP-4) inhibitors have pleiotropic effects on cardiovascular protection beyond the antidiabetic property. However, it remains unknown that the impact of one DPP-4 inhibitor sitagliptin on the survival of mesenchymal stem cells (MSCs) in hypoxia and serum deprivation (H/SD) environment. Methods: The apoptosis and autophagy of MSCs were analyzed in different concentrations of sitagliptin under H/SD condition. For later studies, we tested the relationship between anti-apoptotic and anti-autophagic effects of sitagliptin. The level of cell apoptosis was analyzed by Annexin V-FITC/PI staining, western blot of Bcl-2 and Bax proteins. Autophagy flux was assessed by multiple autophagy related proteins and substrates. Cell autophagy was identified by acridine orange staining, western blot of Beclin 1 and light chain 3 protein, and transmission electron microscopy. Results: We demonstrated that sitagliptin attenuated hypoxia-induced apoptosis and autophagy of MSCs. Furthermore, sitagliptin regulated cell autophagy by Bcl-2/ Beclin 1 pathway in H/SD condition. Conclusions: This study provides insight into the utility of the DPP-4 inhibitor sitagliptin for MSCs transplantation in the ischemic microenvironment that extends its antidiabetic property.

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