Subcellular Fractionation & RNase R Treatment: 3
Purpose: To determine the localization and stability of circ_HECTD1.
Result: Circ_HECTD1 was found mainly in the cytoplasm and resistant to RNase R, indicating its circular nature.
Cell Transfection:
Purpose: To knock down circ_HECTD1 and observe the effects on cell proliferation and apoptosis4.
Result: Knockdown of circ_HECTD1 enhanced cell proliferation and reduced apoptosis in OGD/R-treated HT22 cells5.
EdU & MTT Assays:
Purpose: To assess cell proliferative ability6.
Result: Circ_HECTD1 knockdown increased the number of EdU-positive cells and cell viability, indicating improved cell proliferation.
Flow Cytometry:
Purpose: To analyze cell apoptosis.
Result: Circ_HECTD1 knockdown decreased the apoptosis rate in OGD/R-treated HT22 cells5.
Western Blot:
Purpose: To measure the protein levels related to cell proliferation and apoptosis.
Result: Circ_HECTD1 deficiency led to increased levels of PCNA (a proliferation marker) and Bcl-2 (an anti-apoptotic marker), and decreased levels of Bax and Cleaved PARP (pro-apoptotic markers).
Dual-Luciferase Reporter Assay:1
Purpose: To verify the binding relationship between miR-27a-3p and circ_HECTD1 or FSTL17.
Result: Confirmed that circ_HECTD1 directly interacts with miR-27a-3p and that miR-27a-3p targets FSTL1

RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction):
Purpose: To quantify the expression levels of circ_HECTD1, miR-27a-3p, and FSTL1 mRNA in cells affected by cerebral infarction.
Result: The RT-qPCR results would typically show the relative expression levels of these genes. An increase or decrease in the Ct (Cycle threshold) values would indicate changes in gene expression. For instance, a lower Ct value for circ_HECTD1 might suggest higher expression in cells post-infarction, which could be associated with the cellular response to injury

The article describes several experiments to investigate the role of FSTL1 and miR-125a-3p in adipogenic differentiation and inflammation1. Here’s a summary of the experiments and their results:

Isolation and Differentiation of Preadipocytes: Mouse subcutaneous preadipocytes were isolated and induced to differentiate in vitro2. The study observed morphological changes and lipid accumulation, confirming the suitability of these cells for further experiments.
Plasmid Construction and Luciferase Reporter Assay: A plasmid containing the FSTL1 gene was constructed, and a luciferase reporter assay was performed to validate miR-125a-3p targeting the 3′ UTR region of FSTL134. The assay confirmed that miR-125a-3p could bind to the 3′ UTR of FSTL1, reducing luciferase activity.
Transfection of Plasmids, siRNAs, and miRNAs: The study used transfection techniques to overexpress or silence FSTL1 and miR-125a-3p in preadipocytes5. The results showed that overexpression of FSTL1 inhibited adipogenesis and promoted inflammation, while silencing FSTL1 had the opposite effect67.
Oil Red O Staining and Triglyceride Assay: These assays were used to visualize and quantify lipid accumulation in differentiated adipocytes. Overexpression of FSTL1 reduced lipid droplets, whereas miR-125a-3p promoted lipid accumulation8.
Quantitative Real-Time PCR and Western Blotting: These methods were employed to measure the expression levels of adipogenic and inflammatory genes. The results supported the findings that FSTL1 negatively regulates adipogenesis and promotes inflammation, while miR-125a-3p has the opposite effects6.
Overall, the experiments demonstrated that FSTL1 acts as a pro-inflammatory factor inhibiting adipogenesis, and miR-125a-3p can reverse these effects by targeting FSTL1. This suggests potential therapeutic targets for obesity-related inflammatory diseases.

The article details several experiments conducted to investigate the role of circATP8A1 in gastric cancer progression1. Here’s a summary of the key experiments and their implications:

Exosome Isolation and Characterization: Exosomes were isolated from plasma and characterized to confirm their size and presence of specific markers. This established the foundation for studying the role of exosomal circRNAs in gastric cancer2.
circRNA Microarray Analysis: A microarray was used to identify differentially expressed circRNAs in exosomes from gastric cancer patients compared to healthy individuals. This led to the discovery of circATP8A1 as a novel oncogenic circRNA.
Functional Assays: Various in vitro assays (CCK-8, colony formation, and transwell migration/invasion) were performed to assess the effects of circATP8A1 on gastric cancer cell proliferation, colony formation, and invasiveness. The results indicated that circATP8A1 promotes these malignant behaviors.
Macrophage Polarization: The study explored how exosomal circATP8A1 affects macrophage polarization, finding that it induces M2 polarization through the STAT6 pathway, which is associated with tumor progression3.
Molecular Interactions: The interaction between circATP8A1 and miR-1-3p was investigated using FISH, RIP, RNA pull-down, and luciferase reporter assays5. These experiments confirmed that circATP8A1 acts as a sponge for miR-1-3p, affecting the STAT6 pathway and macrophage polarization4.
Animal Models: In vivo experiments with mice models were conducted to study the effects of circATP8A1 on tumor growth and macrophage polarization, further confirming the oncogenic role of circATP8A1 in gastric cancer progression.

The article describes several experiments conducted to investigate the role of circS100A11 in childhood asthma. Here’s a summary of the key experiments and their implications:

Microarray Analysis:
Purpose: To identify differentially expressed circRNAs in PBMCs of children with asthma compared to healthy controls.
Result: 372 circRNAs were found to be differentially expressed, with circS100A11 being significantly upregulated in asthmatic children.
Macrophage Activation Assays:
Purpose: To determine the effect of circS100A11 on M2a macrophage activation.
Result: circS100A11 facilitated M2a macrophage activation by enhancing the translation of its host gene, S100A11.
Murine Asthma Model:
Purpose: To test the role of circS100A11 in lung inflammation in a cockroach allergen extract (CRE)-induced asthma model.
Result: Overexpression of circS100A11 in macrophages exacerbated lung inflammation, while macrophage-specific knockdown of S100A11 alleviated it.
RNA Immunoprecipitation and Chromatin Immunoprecipitation Assays:
Purpose: To explore the mechanism by which circS100A11 promotes S100A11 translation and affects STAT6 expression.
Result: circS100A11 was found to bind competitively to CAPRIN1, reducing its suppression of S100A11 translation, which in turn enhanced STAT6 expression and M2a macrophage activation.
These experiments collectively suggest that circS100A11 and S100A11 play a significant role in promoting M2a macrophage activation and lung inflammation in asthma, making them potential therapeutic and diagnostic targets.

The article discusses a study on the role of circS100A11 in childhood asthma. Here’s a summary of the experiments and their logic:

Expression Analysis: Researchers identified 372 circRNAs differentially expressed in PBMCs of children with asthma. circS100A11 was notably upregulated and mainly expressed in monocytes.
M2a Macrophage Activation: circS100A11 was shown to facilitate M2a macrophage activation by enhancing the translation of its host gene, S100A11, which worsened lung inflammation in a murine asthma model12.
Mechanistic Insights: The study revealed that circS100A11 competitively binds to CAPRIN1, reducing its suppression on S100A11 translation34. S100A11 then promotes SP3 binding to the STAT6 promoter, enhancing STAT6 expression and M2a macrophage activation56.
Therapeutic Potential: Macrophage-specific knockdown of S100A11 alleviated lung inflammation in the murine model, suggesting circS100A11 and S100A11 as potential therapeutic targets for childhood asthma.
The logic connecting these experiments is to demonstrate how circS100A11 contributes to asthma pathogenesis by regulating macrophage activation and to explore potential therapeutic interventions. The study progresses from identifying a key circRNA to understanding its function, mechanism, and therapeutic implications.

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β-actin, also known as ACTB, is one of the six different actin isoforms identified in humans1. It is a highly conserved protein that plays a crucial role in cell motility, structure, and integrity1. β-actin is a non-muscle cytoskeletal actin, which means it’s involved in forming the cytoskeleton that helps maintain the cell’s shape and provides internal support12.

In addition to its structural role, β-actin participates in various cellular processes such as cell junction assembly, cytoskeleton organization, and membrane organization1. It’s also involved in more complex activities like chromatin remodeling and transcriptional regulation within the nucleus2.

Moreover, β-actin is commonly used as a loading control in Western blotting and as a housekeeping gene standard in quantitative PCR (qPCR) due to its consistent presence across different cell types1. Its molecular weight is approximately 42 kDa1.

In summary, β-actin is a fundamental component of the cellular architecture and is essential for maintaining the cell’s structural integrity and supporting various cellular functions.

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准确来说loading control

The article describes an experiment investigating the role of Follistatin-like protein 1 (FSTL1) in cigarette smoke-induced Chronic Obstructive Pulmonary Disease (COPD). Here’s a summary of the methods and their intended purposes:

Serum and Lung Specimens: To compare FSTL1 and autophagy protein levels between COPD patients and controls.
Animal Models: To observe the effects of chronic cigarette smoke exposure on wild-type mice, FSTL1 ± mice, and FSTL1 flox/+ mice.
3-Methyladenine (3-MA) Treatment: To assess the impact of autophagy inhibition on cigarette smoke-exposed mice.
ELISA and Western Blotting: To measure the expression of FSTL1 and autophagy-associated proteins.
Immunohistochemistry: To visualize the localization and expression of proteins in lung tissues.
Electron Microscopy: To observe autophagosome formation in lung cells.
Bronchoalveolar Lavage Fluid Analysis: To evaluate airway inflammation by measuring cytokines.
Lung Function Tests: To assess the impact of cigarette smoke and FSTL1 on lung function.
The results indicated that FSTL1 promotes cigarette smoke-induced COPD by modulating autophagy, suggesting that targeting FSTL1 and autophagy could be potential therapeutic strategies for treating COPD. The experiments showed increased FSTL1 and autophagy markers in COPD patients and mice exposed to cigarette smoke, and that inhibiting autophagy reduced airway remodeling and inflammation.

COPD Patients vs. Controls:
FSTL1 and Autophagy Biomarkers: Both FSTL1 and autophagy biomarkers were found to be increased in COPD patients compared to controls.
Serum and Lung Specimens: The study observed higher levels of FSTL1 and autophagy-associated protein expression in the serum and lung specimens of COPD patients.
CS-Exposed WT Mice:
Autophagy Activation: There was an upregulation of autophagy activation in CS-exposed mice, accompanied by airway remodeling and inflammation.
3-MA Intervention: CS-exposed WT mice treated with 3-methyladenine (3-MA) showed similar manifestations to CS-exposed FSTL1 ± mice, suggesting that 3-MA could mitigate the effects of CS exposure.
FSTL1 ± Mice:
CS-Induced Autophagy: FSTL1 ± mice exhibited a lower level of CS-induced autophagy compared to control mice.
Resistance to CS-Induced Effects: These mice also resisted CS-induced inflammatory response, airway remodeling, and impaired lung function.
FSTL1 and Autophagy in COPD Pathogenesis:
FSTL1’s Role: The study suggests that FSTL1 promotes CS-induced COPD by modulating autophagy.
Potential Therapeutic Target: Targeting FSTL1 and autophagy could be a new approach to treating cigarette smoke-induced COPD.
Lung Function Assessment:
3-MA and FSTL1 ± Mice: Both 3-MA pretreated WT mice and FSTL1 ± mice showed attenuated lung function decline in response to CS exposure.
Lung Compliance: The study measured lung compliance and total lung capacity, finding that FSTL1 deficiency could protect against the decline of lung function after CS exposure.
These results collectively indicate that FSTL1 plays a significant role in the development of COPD by regulating autophagy, and that manipulating these pathways could offer new therapeutic strategies for COPD. Remember, these are experimental findings and further research is needed to translate these findings into clinical practice.

3-Methyladenine (3-MA) is widely recognized as an inhibitor of autophagy. It functions by blocking the early stages of the autophagy process, particularly the formation of autophagosomes1. Autophagy is a cellular degradation pathway that involves the lysosomal digestion of damaged organelles, proteins, and other cellular components to maintain cellular homeostasis2.

When 3-MA is applied to cells, it inhibits the class III phosphatidylinositol 3-kinase (PI3K), which is essential for the initiation of autophagosome formation1. By preventing the formation of these autophagic vesicles, 3-MA effectively stops the autophagic flux, leading to the accumulation of autophagy substrates such as p62/SQSTM1, which is a protein often used as a marker to study autophagy dynamics1.

In research, 3-MA is commonly used to study the role of autophagy in various physiological and pathological processes by inhibiting this pathway and observing the resulting effects on cells or organisms1. For instance, in the context of neurological diseases, oxidative stress, or atherosclerosis, 3-MA’s inhibition of autophagy can provide insights into how autophagy contributes to these conditions

The article describes a series of experiments to investigate the role of Follistatin-like protein 1 (FSTL1) in asthma. Here’s a summary of the methods and their intended purposes, along with the results:

OVA-Induced Asthma Model: Mice were sensitized and challenged with ovalbumin (OVA) to induce asthma-like symptoms. The control groups received PBS. This model was used to assess the role of FSTL1 in asthma.
Result: Fstl1± mice showed reduced Muc-5AC production and mucus hypersecretion compared to OVA-WT mice1.
Immunohistochemistry & ELISA: These methods were used to detect the protein levels of Muc-5AC, FSTL1, NLRP3, and IL-1β in lung tissues and bronchoalveolar lavage fluid (BALF)2.
Result: Fstl1± mice had decreased levels of these proteins, indicating reduced inflammation.
Western Blot Analysis: This technique was employed to measure the protein expression of NLRP3 and IL-1β in alveolar macrophage cells (MH-S cells)3.
Result: Pretreatment with siFSTL1 or MCC950 reduced the production of NLRP3 and IL-1β induced by OVA or FSTL14.
Cell Culture Experiments: MH-S cells were stimulated with FSTL1 and/or OVA to explore the effect of FSTL1 on the NLRP3/IL-1β signaling pathway.
Result: FSTL1 stimulation increased the expression of NLRP3 and IL-1β, while MCC950 pretreatment inhibited this effect56.
Overall, the study demonstrated that FSTL1 plays a significant role in allergic airway inflammation by activating the NLRP3/IL-1β signaling pathway, and that inhibiting FSTL1 could be a potential therapeutic strategy against asthma78. The experiments collectively aimed to elucidate the mechanisms of FSTL1 in asthma and to explore potential treatments by modulating this protein’s activity.

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The article explains various assays used to assess cell proliferation in the context of papillary thyroid carcinoma (PTC) research:

MTT Assay: This assay measures cell proliferation by adding MTT solution to cells1. MTT is reduced by metabolically active cells, producing a colored formazan product. The intensity of the color, measured by absorbance at 490 nm, correlates with the number of viable cells.
EdU Assay: EdU (5-ethynyl-2’-deoxyuridine) is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis. After labeling cells with EdU, a fluorescent dye is used to detect the incorporated EdU, allowing visualization and quantification of proliferating cells.
Colony Formation Assay: This assay evaluates the ability of a single cell to grow into a colony. Cells are plated at low density and allowed to grow until visible colonies form. The colonies are then fixed, stained, and counted to assess the proliferative potential of the cells.
These assays are utilized in the study to investigate the role of circ_0002111 in PTC cell proliferation and its potential as a therapeutic target2.

Linear PSD3 refers to the linear form of the gene Pleckstrin And Sec7 Domain Containing 3 (PSD3). In the context of this article, PSD3 is the parent gene from which circ_0002111, a circular RNA (circRNA), is derived. The article explains that circ_0002111 is produced from exons 5-9 of the PSD3 gene and has a mature length of 538 base pairs1. Unlike linear RNAs like PSD3, circRNAs like circ_0002111 form a covalently closed continuous loop, which makes them resistant to degradation by RNA exonucleases. This stability allows circRNAs to have regulatory roles in various cellular processes, including acting as microRNA (miRNA) sponges, influencing gene expression, and potentially contributing to diseases such as cancer2. In the case of papillary thyroid carcinoma (PTC), the article discusses the role of circ_0002111 in promoting malignant behaviors by regulating the miR-134-5p/FSTL1 molecular network34. The study uses RT-qPCR to show that linear PSD3 is significantly reduced after RNase R treatment, while circ_0002111 remains stable, highlighting the distinct properties of circRNAs compared to their linear counterparts5.