In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Guidelines for the use and interpretatoin of assays for monitoring autophagy

Metabolites from Bufo gargarizans (Cantor, 1842): A review of traditional uses, pharmacological activity, toxicity and quality control.

Osteosarcoma is an aggressive bone cancer that frequently metastasizes to the lungs. The cytotoxicity of most chemotherapeutics and targeted drugs in the treatment of osteosarcoma is partially lessened. Furthermore, there is a poor response to current chemo- and radiotherapy for both primary lesions and pulmonary metastases of osteosarcoma. There is a clear need to explore promising drug candidates that could improve the efficacy of osteosarcoma treatment. Autophagy, a dynamic and highly conserved catabolic process, has dual roles in promoting cell survival as well as cell death. The role of autophagy has been investigated extensively in different tumor types, and a growing body of research has highlighted the potential value of using autophagy in clinical therapy. Here, we address significant aspects of autophagy in osteosarcoma, including its functions, modulation, and possible therapeutic applications.

/pdf/insight-into-the-role-of-autophagy-in-osteosarcoma-and-its-585jb1unx0.pdf

Insight Into the Role of Autophagy in Osteosarcoma and Its Therapeutic Implication.

Advanced oral squamous cell carcinoma (OSCC) is typically aggressive and closely correlated with disease recurrence and poor survival. Multidrug resistance (MDR) is the most critical problem leading to therapeutic failure. Investigation of novel anticancer candidates targeting multidrug-resistant OSCC cells may provide a basis for developing effective strategies for OSCC treatment. In the present study, we investigated the cytotoxic mechanism of a carbazole alkaloid, namely isomahanine, in a multidrug‑resistant OSCC cell line CLS-354/DX. We demonstrated that CLS-354/DX cells overexpressing multidrug resistance-associated protein 1 (MRP1) were resistant to anticancer drugs cisplatin and camptothecin. Isomahanine effectively induced cytotoxicity against CLS-354/DX cells regardless of resistance. Apoptosis as determined by FITC‑Annexin V/PI staining and western blot analysis of cleaved caspase-3 and cleaved poly(ADP‑ribose) polymerase (PARP) was significantly induced in a time-dependent manner upon isomahanine treatment. Isomahanine-induced caspase‑dependent apoptosis was determined using z-VAD‑fmk. The effects on autophagy in isomahanine-treated cells were investigated via conversion of LC3B and degradation of p62/SQSTM1 (p62). Isomahanine obviously induced autophagic flux as shown by an increase in punctate GFP-LC3B and the LC3B-II/LC3B-I ratio with a concomitant decrease in p62 levels. Autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) protected isomahanine-induced cell death, indicating the activation of autophagic cell death. Endoplasmic reticulum (ER) stress and MAPK activation were examined to elucidate the mechanism underlying cell death. The expression levels of PERK, CHOP and phosphorylated MAPK (p38, ERK1/2 and JNK1/2) were upregulated following isomahanine treatment. We found that p38 MAPK inhibitor (SB203580) significantly attenuated isomahanine-induced apoptosis and autophagic flux and this prevented cell death. Collectively, the present study demonstrated that isomahanine was able to induce ER stress and trigger p38 MAPK-mediated apoptosis and autophagic cell death in multidrug-resistant OSCC cells. The potential cytotoxic action of isomahanine may provide the development of anticancer candidates for treating multidrug-resistant cancer.

/pdf/isomahanine-induces-endoplasmic-reticulum-stress-and-35wis7he03.pdf

Isomahanine induces endoplasmic reticulum stress and simultaneously triggers p38 MAPK-mediated apoptosis and autophagy in multidrug-resistant human oral squamous cell carcinoma cells

In the present study, the components of A. cinnamomea (AC) mycelia were systematically analyzed. Subsequently, its hepatoprotective effects and the underlying mechanisms were explored using a mouse model of acute alcohol-induced liver injury. AC contained 25 types of fatty acid, 16 types of amino acid, 3 types of nucleotide, and 8 types of mineral. The hepatoprotective effects were observed after 2 weeks of AC treatment at doses of 75 mg/kg, 225 mg/kg, and 675 mg/kg in the mouse model. These effects were indicated by the changes in the levels of aspartate aminotransferase, alanine aminotransferase, several oxidation-related factors, and inflammatory cytokines in serum and/or liver samples. AC reduced the incidence rate of necrosis, inflammatory infiltration, fatty droplets formation, and cell apoptosis in liver detecting via histological and TUNEL assay. In addition, AC reduced the expression of cleaved caspase-3, -8, and -9 and the levels of phosphor-protein kinase B (Akt) and phosphor-nuclear factor-κB (NF-κB) in the liver samples. Collectively, AC-mediated hepatoprotective effects in a mouse model of acute alcohol-induced liver injury are the result of reduction in oxidative stress. This may be associated with Akt/NF-κB signaling. These results provide valuable evidence to support the use of A. cinnamomea as a functional food and/or medicine.

/pdf/hepatoprotective-effects-of-antrodia-cinnamomea-the-ofaek0i8rp.pdf

Hepatoprotective Effects of Antrodia cinnamomea: The Modulation of Oxidative Stress Signaling in a Mouse Model of Alcohol-Induced Acute Liver Injury

The main objective of this study was to explore whether autophagy could be triggered by cinobufagin, and to clarify the role of autophagy in the antitumor effects of cinobufagin on U2OS cells and the underlying mechanisms. U2OS cells were exposed to 15, 30, 60 and 120 mg/l cinobufagin for 0, 12, 24 and 48 h. An MTT assay was used to measure cell viability. FITC-Annexin Ⅴ/PI staining and flow cytometry were used to analyze the apoptotic ratio, while apoptotic morphological changes were assessed by PI and Hoechst 33258 viable cell staining. The effects of autophagy on the cells were investigated with GFP-LC3b green fluorescence plasmid transfection and transmission electron microscopy. The levels of caspase-3, -8, - 9, cleaved PARP, LC3-II/LC3-I, p62 and the activation of JNK/p-38 were detected by western blot analysis. Reactive oxygen species (ROS) fluorescence intensity was examined under fluorescence microscopy with an analysis software system. Cell proliferation was obviously inhibited by cinobufagin in a dose- and time-dependent manner. The apoptosis ratio was gradually increased with treatment time as evidenced by flow cytometric analysis and Hoechst 33258 staining. Exposure to cinobufagin resulted in the activation of caspase-3, -8, -9, as well as cleaved PARP which indicated that cinobufagin induced caspase-dependent apoptosis. Autophagy was confirmed in the cinobufagin-treated cells as evidenced by formation of autophagosomes, accumulation of GFP-LC3 fluorescence particles as well as the upregulation of LC3-II/LC3-I levels. Inhibition of autophagy diminished apoptosis as detected by the MTT assays. Moreover the percentage of apoptotic cells decreased following pretreatment with 3-MA, CQ and si-beclin-1. Cinobufagin also induced phosphorylation of the JNK and p38 signaling pathway as well as ROS generation. The JNK and p38 inhibitors significantly attenuated coexistence of apoptosis and autophagy-related proteins. The ROS scavenger also prevented phosphorylation of the JNK and p38 signaling pathway. Our research proved that cinobufagin triggered apoptosis and autophagic cell death via activation of the ROS/JNK/p-38 axis.

/pdf/cinobufagin-induces-autophagy-mediated-cell-death-in-human-3tlgsr0vvo.pdf

Cinobufagin induces autophagy-mediated cell death in human osteosarcoma U2OS cells through the ROS/JNK/p38 signaling pathway

Kinsenoside (KD) exhibits anti-inflammatory and immunosuppressive effects. Dendritic cells (DCs) are critical regulators of the pathologic inflammatory milieu in liver fibrosis (LF). Herein, we explored whether and how KD repressed development of LF via DC regulation and verified the pathway involved in the process. Given our analysis, both KD and adoptive transfer of KD-conditioned DCs conspicuously reduced hepatic histopathological damage, proinflammatory cytokine release and extracellular matrix deposition in CCl4-induced LF mice. Of note, KD restrained the LF-driven rise in CD86, MHC-II, and CCR7 levels and, simultaneously, upregulated PD-L1 expression on DCs specifically, which blocked CD8+T cell activation. Additionally, KD reduced DC glycolysis, maintained DCs immature, accompanied by IL-12 decrease in DCs. Inhibiting DC function by KD disturbed the communication of DCs and HSCs with the expression or secretion of α-SMA and Col-I declined in the liver. Mechanistically, KD suppressed the phosphorylation of PI3K-AKT driven by LF or PI3K agonist, followed by enhanced nuclear transport of FoxO1 and upregulated interaction of FoxO1 with the PD-L1 promoter in DCs. PI3K inhibitor or si-IL-12 acting on DC could relieve LF, HSC activation and diminish the effect of KD. In conclusion, KD suppressed DC maturation with promoted PD-L1 expression via PI3K-AKT-FoxO1 and decreased IL-12 secretion, which blocked activation of CD8+T cells and HSCs, thereby alleviating liver injury and fibro-inflammation in LF. 

http://manuscript.elsevier.com/S1043661822000378/pdf/S1043661822000378.pdf

Kinsenoside attenuates liver fibro-inflammation by suppressing dendritic cells via the PI3K-AKT-FoxO1 pathway

The aim of the present study was to ascertain whether or not autophagy is induced by tanshinone IIA (TanIIA), and to explore the crosstalk between autophagy and apoptosis in regards to the antitumor effects of TanIIA on MG-63 cells and the potential mechanism. MG-63 cells were cultured in vitro with various concentrations of TanIIA (0, 2.5, 5, 10 and 20 mg/l) for 0, 24, 48 and 72 h, respec- tively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay was used to evaluate the inhibition of the proliferation of osteosarcoma MG-63 cells by TanIIA or in the presence/absence of chloroquine (CQ). Autophagic vacuoles and characteristic autophagosomes were observed by transmission electron microscopy (TEM). TanIIA-induced autophagy in MG-63 cells was confirmed by GFP-LC3 punc - tate fluorescence. The expression levels of apoptosis-related proteins caspase-3, caspase-8, caspase-9 and cleaved-PARP and autophagy-related proteins LC3II/LC3I and Beclin-1 were detected by western blotting. FITC-Annexin V/propidium iodide (PI) staining, flow cytometry and Hoechst 33258 staining were used to analyze the apoptotic rate. Fluorescence intensity of reactive oxygen species (ROS) was examined under a fluorescence microscope using an analysis software system. Cell proliferation was obviously inhibited by TanIIA in a dose- and time-dependent manner. Generation of autophagy was triggered by TanIIA (0-20 mg/l) treatment, and in a Beclin-1-dependent manner. Compared with the control group, the apoptosis ratio following treatment with 2.5 mg/l TanIIA failed to achieve statistical significance. Expression of caspase-3, -8 and -9, and cleaved-PARP in the other groups was gradually enhanced in dose-dependent manner. Our analysis also suggested that the influence of autophagy on TanIIA cyto - toxicity had a phase effect; with low -dose drugs and shorter treatment periods, autophagy functioned as a damage repair mechanism. In conrast, when the cells were treated with higher doses of TanIIA for longer treatment periods, autophagic cell death contributed to apoptosis. Furthermore, generation of ROS occurred in a dose-dependent manner and pretreatment with NAC, a selective ROS scavenger, blocked the coexistence of Beclin-1 autophagy and caspase-dependent apoptosis. In conclusion, our findings provide strong evidence that TanIIA may be a potential therapeutic drug against osteosarcoma. Moreover, its cytotoxity can be enhanced with ROS agonists.

/pdf/crosstalk-between-beclin-1-dependent-autophagy-and-caspase-9egq3hy6ag.pdf

Crosstalk between Beclin-1-dependent autophagy and caspase‑dependent apoptosis induced by tanshinone IIA in human osteosarcoma MG-63 cells

Studies have reported a relationship between human epidermal growth factor receptor 4 (HER4), a ubiquitously expressed and unique member of the ErbB family, and clinicopathological features of osteosarcoma. However, further investigation is warranted. HER4 expression was analyzed by quantitative reverse transcription-polymerase chain reaction, western blotting, and immunohistochemistry. The relationship between HER4 expression and the prognosis of patients with osteosarcoma was determined by constructing a Kaplan-Meier curve. Cell viability and proliferation were investigated by MTT and colony formation assays. The mechanism underlying HER4-modulated proliferation and invasion/migration of osteosarcoma cells was determined by short hairpin RNA (shRNA) interference, colony formation, migration, invasion, and western blotting experiments. Spheroid formation assay and CD133+ cell populations were used to examine HER4-induced stem-like traits. The present findings revealed that HER4 was overexpressed in both osteosarcoma cells and tissues. Moreover, this overexpression was associated with high Enneking stage, metastasis, and recurrence. Sh-HER4 showed obviously suppressed cell viability, colony formation, and invasion/migration. In addition, knockdown of HER4 markedly attenuated the spheroid size and proportion of CD133-positive cells, as well as the expression of stemness markers. Sh-HER4 also reduced the tumor size, downregulated the expression of phosphorylated-PI3K (p-PI3K) and p-AKT, and increased that of p-phosphatase and tensin homolog (p-PTEN) in mouse tissue. From a mechanistic perspective, HER4 knockdown activated p-PTEN and suppressed p-PI3K and p-AKT expression. HER4 promoted osteosarcoma progression through inactivation of the PTEN-PI3K/AKT pathway. Taken together, the results indicate that HER4 represents a novel target in osteosarcoma progression and stemness modulation, and may be of value for the development of treatments against osteosarcoma.

/pdf/her4-promotes-osteosarcoma-progression-and-predicts-poor-2ngde075.pdf

HER4 Promotes Osteosarcoma Progression and Predicts Poor Prognosis through the PTEN-PI3K/AKT Pathway

OBJECTIVE
To express matrix remodeling associated 7 (MXRA7) in the human acute myeloid leukemia SHI-1 cell line and to assess the role of MXRA7 in the biological function of SHI-1 cells.


METHODS
The full-length cDNA sequence of human MXRA7 was synthesized and subcloned into the lentivirus shuttle vector pRRL-Venus. SHI-1 cells were transfected with the lentivirus which was packaged with 293T cells. The YFP-positive cells were sorted by flow cytometry and the stable cell lines were obtained by expanded culture. The expression and distribution of MXRA7 in SHI-1 cells were verified by real-time qPCR, Western blot and laser confocal techniques. Cell proliferation and cell cycle were measured by flow cytometry, and apoptosis was determined by Annexin V and 7-AAD staining. The expression of apoptosis related proteins were detected by Western blot.


RESULTS
The stable SHI-1 cell line overexpressing MXRA7 was established successfully. Laser confocal analysis confirmed that MXRA7 was expressed in the cytoplasm of SHI-1 cells. Compared with the control cell line, the overexpression of MXRA7 showed no effect on the cell proliferation and cell cycle, but reduced the percentage of apoptosis cells induced by methotrexate. Moreover, the expression of BCL-2 protein was increased by overexpression of MXRA7, which can inhibit cell apoptosis.


CONCLUSION
The SHI-1 stable cell line overexpressing MXRA7 was established successfully, and MXRA7 could inhibit drug-induced apoptosis through increasing the expression of BCL-2 protein.

[The Effect of Matrix Remodeling Associated 7 (MXRA7) Expression on the Biological Function of SHI-1 Cells].

Handle everyday research tasks with reliable, citation-backed results

Your personal Research Agent to handle research tasks with citation-backed results

Popular Tasks used by Researchers

How can I help with your research?

Meet SciSpace

Get more enhanced response by uploading the PDFs you want me to reference.

No relevant PDFs in your library

SciSpace is the AI research assistant for academics. Run systematic literature reviews on 280M+ papers, and write papers with cited sources. Trusted by 1M+ students, PhDs & researchers.

SciSpace | AI for Research

Analyze PDFs

Code & Manuscripts

Funding & Grants

Literature & Patents

Medical & Clinical Data

Systematic Review

Visualize & Present

Web & Data

Build a Google Scholar-like website for your research.

Build a website

Create charts and images for your research

Create a Chart

Write a paper for submission to a journal

Draft a manuscript

Patent Search

Design eye-catching scientific posters in minutes.

Scientific Poster Generation

Systematic Literature Review

One task is running at the moment. Your messages will be shown right after.

Drag and drop or click here to browse

Loved by <highlight>1 million+</highlight> researchers

Extract a list of specific topics and their sources from unstructured text

Topics

Compare and analyze relevant papers that matches with your search

Papers

Get insights from PDFs and bookmarked papers from your library

My library

Recent searches

Try searching for:

Catch AI-generated content in scholarly and non-scholarly content

{ai} Detector

Ai Writer

Get PDF Summaries, highlighted text explanations 

Chat with PDF

Effortlessly create in-text citations and bibliographies in APA and 2,500 other formats

Citation generator

Get explanations, summaries, and answers on academic papers

Ease up your research workflow with {scispace}'s cohort of exciting AI tools

Elevate your academic writing skills and convey your ideas the way you want

Paraphraser

Explore our range of reading and writing tools

Your file is being prepared and should be ready in a few minutes. If it's a large file, it might take a bit longer. You can close this window, and we'll email you the file when it's done.

You have reached a maximum limit of <strong>{limit}</strong> columns in the table. Remove at least <strong>1</strong> column to add or create another one.

Kun Ma

Author Tools

Chat about Author