The current study was granted by &#34;Study on the key issues of curative effect of Koumiss on regional diseases of Mongolian medicine&#34; in 2018 Supported Project of the science and technology program of the Department of Science and Technology of Inner Mongolia Autonomous Region.

NOTE: See the Table of Materials for details related to all materials, instruments, and reagents used in this protocol.
1. Cell culture
<ol>
	<li>Culture HepG2 cells in culture flasks containing Dulbecco&#39;s Modified Eagle Medium (DMEM) (containing 10% fetal bovine serum [FBS], 100 units/mL penicillin, and 100 &#181;g/mL streptomycin). Maintain the culture flasks at 37 &#176;C in a 5% CO2 incubator.</li>
</ol>
2. Effect of oleic acid on cell viability as measured by cell counting kit-8
<ol>
	<li>Dissolve a specific volume of OA in dimethyl sulfoxide (DMSO) to achieve a concentration of 200 mM. Store the solution at -20 &#176;C for future use.</li>
	<li>Seed HepG2 cells in a 96-well plate at a cell density of 6 &#215; 103 cells per well. Add 100 &#181;L of DMEM to each well. Incubate and culture the cells at 37 &#176;C in a 5% CO2 incubator for 24 h. Divide the HepG2 cells into two groups:
	<ol>
		<li>Control group: add cell culture medium.</li>
		<li>OA group: add OA to the cell culture medium to achieve final concentrations of 0.125 mM, 0.25 mM, 0.5 mM, and 1 mM.</li>
	</ol>
	</li>
	<li>After 24 h of initial incubation, discard the supernatant from each well and add OA according to the specified grouping, adding 100 &#181;L per well. For the control group, add 100 &#181;L of cell culture medium to each well. Continue to incubate the cells for another 24 h. 
	NOTE: Ensure each group has six replicate wells. To prevent evaporation, add 100 &#181;L of phosphate-buffered saline (PBS) to the outer ring of wells in the 96-well plate.</li>
	<li>After 24 h of incubation, add 10 &#181;L of cell counting kit-8 (CCK-8) to each well, mix gently, and incubate for 2 h in the dark. Remove the 96-well plate from the incubator, place it in the microplate reader, and measure the absorbance value at 450 nm (A450). The GI50 values were counted according to the OD.</li>
</ol>
3. Oil Red O staining to observe intracellular lipid droplet formation
<ol>
	<li>Seed HepG2 cells in 6-well cell culture plates at a density of 5 &#215; 105 cells per well and culture the plates in a constant-temperature incubator for 24 h. Refer to Figure 1 for a visual representation of the described steps.</li>
	<li>After 24 h of cell culture, add 2 mL of cell culture medium containing OA to each well, achieving final concentrations of 0.125 mM, 0.25 mM, 0.5 mM, and 1 mM. After an additional 24 h, remove the cell culture medium from each well and wash twice with PBS. Add 1 mL of ORO fixative to each well and incubate for 30 min.</li>
	<li>Prepare the ORO staining solution by mixing staining solution A with staining solution B at a ratio of 3:2. Allow the mixture to stand at room temperature for 10 min, then filter it once through a 0.45 &#181;m filter. Store the filtered solution in a centrifuge tube protected from light until use.</li>
	<li>Discard the fixative and wash twice with distilled water. Add 1 mL of 60% isopropanol to each well and incubate for 30 s. Discard 60% of the isopropanol solution and add 1 mL of freshly prepared ORO stain solution to each well before incubating for 20 min. Discard the ORO staining solution, add 1 mL of 60% isopropanol to each well, and incubate for 30 s. Wash 5x with water to remove excess dye.</li>
	<li>Cover the cells with distilled water and observe under a microscope. Once the images are collected, discard the liquid in the plate and allow it to dry. Then, add 2 mL of isopropanol to each well and shake the plate on an orbital shaker for 10 min. Transfer the liquid to a new 96-well plate, with 16 wells in each group, adding 100 &#181;L per well. Calculate the lipid content by measuring the optical density (OD) of each well using a microplate reader at 510 nm (A510).</li>
</ol>
4. Effects of different concentrations of oleic acid on total triglyceride in HepG2 cell supernatant
<ol>
	<li>Equilibrate the kit at room temperature for 20 min and prepare the required plates for the experiment.</li>
	<li>Collect the cell supernatant and centrifuge at 1,570 &#215; g for 10 min. Set standard wells and testing sample wells. Add 50 &#181;L of standard ([S0 &#8594; S5] concentration followed by: 0, 0.5, 1, 2, 4, 8 mmol/L) to standard wells. In addition to the blank and standard wells, add 10 &#181;L of different samples to the sample wells, followed by adding 40 &#181;L of sample diluent to each well. Add 100 &#181;L of detection antibody-horseradish peroxidase to each well, seal with a plate membrane, and incubate at 37 &#176;C for 1 h in a constant temperature oven.</li>
	<li>Discard the supernatant, blot dry on dust-free paper, and wash each well with 1x washing solution. Leave to stand at room temperature for 1 min. Repeat the washing process 5x.</li>
	<li>Add 50 &#181;L of substrate A and 50 &#181;L of substrate B to each well. Mix gently and incubate for 15 min at 37 &#176;C. Add 50 &#181;L of termination solution to each well and measure the OD value of each well at 450 nm (A450) within 15 min.</li>
	<li>Plot the concentration of the standard along the x-axis and the corresponding absorbance (OD) value along the y-axis to perform linear regression and derive the curve equation to calculate the concentration value of each sample.</li>
</ol>
5. Statistical analysis
<ol>
	<li>Determine significant differences in quantitative data.</li>
	<li>Calculate the mean &#177; standard deviation (SD) and graphically represent the data. Consider P &#60; 0.05 to be statistically significant.</li>
</ol>

Oleic Acid-Induced HepG2 Cells for Metabolic Disease Analysis 

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The authors declare no conflicts of interest.

MASLD is a clinicopathological syndrome characterized by excessive intracellular fat deposition in hepatocytes due to factors beyond alcohol and other established liver-damaging agents18. MASLD is intricately linked to acquired metabolic stress liver injury, notably associated with insulin resistance and genetic susceptibility. To effectively study and screen drugs for MASLD, it is crucial to select an appropriate experimental model. Establishing a cell model is particularly vital in MASLD researc...

Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a range of conditions, including simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma1,2,3,4,5,6, all attributed to factors other than alcohol consuption7. MASLD is the most prevalent liver disease caused by metabolic liver injury, affecting nearly one-quarter of the global population8,9,10,11,12. While the precise pathogenesis of MASLD has not yet been elucidated, various theories attempt to explain its development. One prevailing notion suggests a departure from the classic &#34;two-hit&#34; theory towards a &#34;multiple-hit&#34; model1. Central to these hypotheses is the role of insulin resistance, which is believed to be pivotal in MASLD pathogenesis13. Research indicates that insulin resistance in hepatocytes leads to increased levels of free fatty acids, subsequently forming triglycerides stored within the liver14,15.
Researchers have used both in vivo and in vitro models to simulate fat deposition in MASLD; yet fully replicating its pathomechanism remains challenging. Despite this limitation, these models have been instrumental in studying potential therapeutic targets for MASLD. However, the development of a stable model of MASLD is crucial. While animal models are effective, they are time-consuming and expensive, thus highlighting the growing interest in in vitro cellular models. These models often use single or multiple free fatty acids such as oleic acid (OA) and palmitic acid to recreate diet-induced MASLD. Among these, the human hepatoblastoma cell line HepG2 is often used to establish in vitro cellular models of MASLD.
OA induction stimulates HepG2 cells to replicate fatty deposition akin to MASLD, a method with a well-established history. The aim of this study was to demonstrate the viability, Oil Red O (ORO) staining, lipid content, and triglyceride (TG) level of HepG2 cells treated with 0.25 mM OA. The objective of this experiment was to provide further evidence for the development of MAFLD modeling studies.

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	<tbody>
		<tr>
			<td>0.22 &#181;m filter</td>
			<td>Millex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA (1x) Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#181;m filter</td>
			<td>Millex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Crygenic Vials</td>
			<td>CORNING</td>
			<td>430659</td>
			<td></td>
		</tr>
		<tr>
			<td>25 cm2 Cell Culture Flask</td>
			<td>CORNING</td>
			<td>430639</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plate</td>
			<td>CORNING</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well cell culture plate</td>
			<td>CORNING</td>
			<td>3599</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Count Plate</td>
			<td>Shanghai Jing Jing Biochemical Reagent &#38; Instrument Co.</td>
			<td>02270113</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counting Kit-8 assays</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>CA1210-1000T</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>NUAIRE</td>
			<td>NU-5710E</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;DMSO Dimethyl sulfoxide&#160;</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>D8371</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>8122691</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzyme Labeling Equipment</td>
			<td>Tecan</td>
			<td>Spark</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Qualified</td>
			<td>Gibco</td>
			<td>10099141</td>
			<td></td>
		</tr>
		<tr>
			<td>HepG2 cells line</td>
			<td>Beijing North China Chuanglian Biotechnology Research Institute (BNCC)</td>
			<td>221031</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Triglyceride (TG) ELISA instruction</td>
			<td>Nanjing Jiacheng Bioengineering Institute</td>
			<td>20170301</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope for Cell Culture</td>
			<td>Leica</td>
			<td>DMi1&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Tianjin Zhiyuan Chemical Reagent Co.</td>
			<td>2021030141</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red Stain Kit, For Cultured Cells</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>G1262</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic acid&#160;</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>A502071</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS 24.0</td>
			<td></td>
			<td>Statistics software</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro modeling of fat deposition in metabolic dysfunction-associated steatotic liver disease

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading to significant health challenges. Previous reports have studied the establishment of animal and cellular models for MASLD, highlighting differences between them. In this study, a cellular model was created by inducing fat accumulation in MASLD. HepG2 cells were stimulated with the unsaturated fatty acid oleic acid at various concentrations (0.125 mM, 0.25 mM, 0.5 mM, 1 mM) to emulate MASLD. The model&#39;s efficacy was assessed using cell counting kit-8 assays, Oil Red O staining, and lipid content analysis. This study aimed to create a simple-to-operate cellular model for MASLD cells. Results from the cell counting kit-8 assays showed that the survival of HepG2 cells was dependent on the concentration of oleic acid, with a GI50 of 1.875 mM. Cell viability in the 0.5 mM and 1 mM groups were significantly lower than those in the control group (P &#60; 0.05). Furthermore, Oil Red O staining and lipid content analysis examined fat deposition at varying oleic acid concentrations (0.125 mM, 0.25 mM, 0.5 mM, 1 mM) on HepG2 cells. The lipid content of the 0.25 mM, 0.5 mM, and 1 mM groups was significantly higher than that of the control group (P &#60; 0.05). Additionally, triglyceride levels in the OA groups were significantly higher than those in the control group (P &#60; 0.05).

Effect of oleic acid on cell viability 
HepG2 cells were exposed to varying concentrations of OA (0 mM, 0.125 mM, 0.25 mM, 0.5 mM, 1 mM), resulting in a decrease in cell survival rates at 0.125 mM, 0.25 mM, 0.5 mM, and 1 mM compared to 0 mM. Statistical significance was observed at 0.5 mM (P &#60; 0.05) and 1 mM (P &#60; 0.05) when compared to 0 mM. The results of OA&#39;s impact on cell viability, as assessed by the CCK-8 kit, are shown in Fig...

Author Spotlight: Establishing MASLD Cell Models for Investigating Disease Mechanisms and the Lipid-Lowering Effects of Koumiss

This article describes the use of oleic acid-induced HepG2 cells as a model for metabolic dysfunction-associated steatotic liver disease.

In Vitro Modeling of Fat Deposition in Metabolic Dysfunction-Associated Steatotic Liver Disease

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading ...

in-vitro-modeling-fat-deposition-metabolic-dysfunction-associated

Research

JoVE Journal

Biology

611 Views.  Inner Mongolia Medical University. This article describes the use of oleic acid-induced HepG2 cells as a model for metabolic dysfunction-associated steatotic liver disease.

Video: Author Spotlight: Establishing MASLD Cell Models for Investigating Disease Mechanisms and the Lipid-Lowering Effects of Koumiss

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is often not detected until drugs have reached clinical stages, risking patients' safety and leading to substantial losses for the pharma industry. Taking into account that standard 2D models have limitations in detecting DILI it is essential to develop in vitro models that are more predictive to improve data translatability. To understand causality and mechanistic aspects of DILI in detail, a human liver MPS consisting of human primary liver parenchymal and non-parenchymal cells (NPCs) and cultured in 3D microtissues on an engineered scaffold under perfusion has been developed. Cryopreserved primary human hepatocytes (PHHs) and Kupffer cells (HKCs) were cocultured as microtissues in the MPS platform for up to two weeks, and each compound of interest was repeatably dosed onto liver microtissues at seven test concentrations for up to four days. Functional liver-specific endpoints were analyzed (including clinical biomarkers such as alanine aminotransferase, ALT) to evaluate liver function. Acute and chronic exposure to compounds of various DILI severities can be assessed by comparing responses to single and multi-dosed microtissues. The methodology has been validated with a broad set of severe and mildly hepatotoxic compounds. Here we show the data for pioglitazone and troglitazone, well-known hepatotoxic compounds withdrawn from the market for causing liver failures. Overall, it has been shown that the liver MPS model can be a useful tool to assess DILI and its association with changes in hepatic function. The model can additionally be used to assess how novel compounds behave in distinct patient subsets and how toxicity profiles may be affected by liver disease states (e.g., viral hepatitis, fatty liver disease).

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

Drug-induced liver injury (DILI) is a major cause of drug failure. A protocol has been developed to accurately predict the DILI liability of a compound using a liver microphysiological system (MPS). The liver model uses the coculture of primary hepatic cells and translationally relevant endpoints to assess cellular responses to treatment.

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is ...