Skip to main content
NCBI home page
Redox Report : Communications in Free Radical Research logoLink to Redox Report : Communications in Free Radical Research
. 2023 Sep 26;28(1):2260646. doi: 10.1080/13510002.2023.2260646

Slc7a11 stimulates glutathione synthesis to preserve fatty acid metabolism in primary hepatocytes

Yifan Liu 1,*, Kaimin Wu 1,*, Yinkun Fu 1, Wenyan Li 1,CONTACT, Xu-Yun Zhao 1,CONTACT
PMCID: PMC10540662  PMID: 37750478

ABSTRACT

Primary hepatocytes are widely used as a tool for studying metabolic function and regulation in the liver. However, the metabolic properties of primary hepatocytes are gradually lost after isolation. Here, we illustrated that fatty acid metabolism is the major compromised metabolic process in isolated primary hepatocytes, along with drastically decreased GSH and ROS content, while lipid peroxidation is increased. Gain- and loss-of-function studies revealed that Slc7a11 expression is critical in maintaining fatty acid metabolism and facilitating hormone-induced fatty acid metabolic events, which is synergistic with dexamethasone treatment. Intriguingly, Slc7a11 expression and dexamethasone treatment cooperatively upregulated AKT and AMPK signaling and mitochondrial complex expression in primary hepatocytes. Furthermore, direct treatment with reduced GSH or inhibition of ferroptosis is sufficient to drive protective effects on fatty acid metabolism in primary hepatocytes. Our results demonstrate that Slc7a11 expression in isolated primary hepatocytes induces GSH production, which protects against ferroptosis, to increase fatty acid metabolic gene expression, AKT and AMPK signaling and mitochondrial function in synergy with dexamethasone treatment, thereby efficiently preserving primary hepatocyte metabolic signatures, thus providing a promising approach to better reserve primary hepatocyte metabolic activities after isolation to potentially improve the understanding of liver biological functions from studies using primary hepatocytes.

KEYWORDS: Slc7a11, GSH, ferroptosis, primary hepatocytes, fatty acid metabolism, dexamethasone, AKT/AMPK signaling‌, mitochondrial homeostasis

Introduction

The liver is a vital metabolic organ in the body that modulates glucose, fatty acid and amino acid metabolism for whole-body metabolic homeostasis. The loss of liver function is a leading cause of metabolic diseases such as NAFLD and diabetes. Hepatocytes are the major cell type in the liver and play the most important role in liver function. Hepatocytes in culture have been widely used for decades as an essential tool to address liver metabolic functions, detoxification of toxicants and key regulation at the molecular level [1]. Hepatocytes in culture are obtained from a variety of different sources, such as hepatocellular carcinoma and embryonic stem cells, and primary hepatocytes, due to their direct isolation and extraction from the liver, are the closest model to the hepatocyte phenotype in vivo, especially as their plasma membrane maintains an active uptake/secretion of nutrients for metabolic processes [2–7]. As such, primary hepatocytes are considered the gold standard for in vitro hepatocyte culture models and play an extensive and irreplaceable role in scientific research [8].

Although primary hepatocytes have been the closest model to in vivo cells, isolating and culturing primary hepatocytes results in transcriptional reprograming and loss of certain metabolic functions [9]. During the isolation process of two-step collagenase perfusion, nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) mediate the destruction of liver tissue integrity and the occurrence of ischemia-reperfusion injury, activate inflammatory and proliferative responses [10, 11] and suppress liver-specific gene expression [12, 13]. Changes in cellular mRNA levels can be detected after just a few hours of in vitro cell culture [14, 15]. Proteomic analysis of isolated primary hepatocytes revealed an increasing number of significantly and differentially expressed proteins observed at 24, 72 and 168 h compared to freshly isolated cells [16]. To counteract the process of functional loss during primary hepatocyte isolation and culture, some strategies have been developed to maintain the characteristics of hepatocytes. The classical approach to balance hepatocyte loss of function is to mimic the natural hepatocyte microenvironment in an artificial in vitro environment designed to maintain hepatic gene expression in the in vivo state, with dexamethasone (dex) being one of the most commonly used hormone-based additives to hepatocyte culture media [9, 17, 18]. In addition, attempts have been made to use methods such as 3D cell culture and the use of various small molecule inhibitors [8, 9, 19], which are either limited by their incomplete maintenance of hepatocyte properties or are more complex to perform and have not been widely used in primary hepatocyte culture [20]. Directly targeting the key regulation of major metabolic processes, which causes the loss-of-function of primary hepatocytes, may provide a novel way to counteract the loss of identity of primary hepatocytes in culture.

The Xc-system is a sodium-independent reverse transporter of cystine and glutamate in the cell membrane that transports extracellular cystine into the cell in a 1:1 ratio while transporting intracellular glutamate to the outside of the cell [21]. It consists of two subunits, the light chain subunit SLC7A11 and the heavy chain subunit SLC3A2, of which SLC7A11 is a 12-fold transmembrane protein that is highly specific for cystine and glutamate and responsible for the major transport activity [22, 23]. The expression of Slc7a11 seems to be rather limited in vivo, with lymphoid organs and the central nervous system being the main tissues constituting the expression of the Xc-system [24]. In addition, Slc7a11 expression is induced in many cells cultured in vitro [25]. The most obvious function of Slc7a11 in vitro is to provide cystine to the cell, which is subsequently reduced to cysteine in the cell for glutathione (GSH) synthesis [26].

GSH is a tripeptide composed of glutamate, cysteine and glycine, which is the most important low molecular weight antioxidant synthesized and ubiquitously present in cells [27]. The sulfhydryl group (-SH) of cysteine in glutathione performs its main function in reduction reactions, as it is easily oxidized and dehydrogenated [28]. GSH is one of the main physiological free radical scavengers that can effectively scavenge excess reactive oxygen species (ROS) and endogenous and exogenous electrophilic substances and plays a role in maintaining intracellular redox balance, reducing oxidative damage and preventing apoptosis [29]. It plays a role in maintaining intracellular redox balance, reducing oxidative damage and preventing apoptosis [30]. Various studies have shown that oxidative stress is a major cause of various liver diseases. Disruption of GSH homeostasis is documented in many liver diseases, such as chronic alcoholic liver disease, hepatitis C infection, hypoxia/ischemia and/or reperfusion injury and nonalcoholic fatty liver disease (NAFLD) [31–34]. In addition, GSH can serve as a hydrogen donor used by glutathione peroxidase 4 (Gpx4) to reduce lipid hydroperoxides. Recent studies have demonstrated that a decrease in intracellular GSH levels leads to cell ferroptosis, a form of iron-dependent cell death characterized by overwhelming accumulation of membrane lipid peroxides [35].

Here, we report that fatty acid metabolism is the most disrupted metabolic process in primary hepatocytes. Manipulation of the expression of Slc7a11 in the mouse liver influences GSH content, which governs fatty acid metabolic activities in primary hepatocytes. Finally, we demonstrated that GSH supplementation and ferroptosis inhibition largely preserve fatty acid metabolic characteristics in primary hepatocytes, which may be a novel strategy for maintaining primary hepatocyte identity.

Materials and methods

Mice

All animal studies were performed according to procedures approved by the University Committee on Use and Care of Animals at Shanghai Jiaotong University. Mice were maintained in 12/12 h light/dark cycles and fed regular rodent chow. Wild-type C57BL/6J mice (JAX stock 000664) were purchased from the JAX lab, and Slc7a11 flox/flox mice were generated by Gempharmatech. For AAV transduction, we injected approximately 1 × 1011 genome copies of AAV vectors per mouse via the tail vein.

Isolation and culture of primary hepatocytes

Primary hepatocytes were isolated using a two-step collagenase perfusion method. After mice were euthanized, the livers were perfused with perfusion buffer and collagenase successively through the right atrium. The moderately digested liver was minced, centrifuged and washed twice, and then the cells were spread in a culture dish coated with collagen. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in an incubator at 37°C in a 5% CO2 atmosphere.

AAV virus packaging and purification

Purification of adeno-associated viruses requires steps such as virus packaging, virus purification, and titer determination. Packaging of adeno-associated viruses requires three types of plasmids: (1) vector plasmids containing the target gene; (2) pAdDeltaF6 (DF6) providing E2a, E4, and VARNA; and (3) different serotype plasmids depending on the affinity of the organ. In this experiment, we used the liver-specific serotype AAV2/8. 293 T cells were transfected with the three plasmids for 60 h, and the precipitates were collected. Viral solutions with high titers were obtained after virus concentration, density gradient centrifugation, and column purification steps.

Cell culture

Primary hepatocytes were isolated as previously described by using collagenase type II from C57BL/6J mice [36]. Hepatocytes were maintained in DMEM containing 10% FBS and 1% penicillin streptomycin (BasalMedia, S110) at 37°C and 5% CO2. A total of 3 × 105 cells were seeded in each well of a 12-well plate after isolation. After attachment, the cells were immediately treated with vehicle (DMSO) or Dex for 24 h. The cell culture experiments were performed in triplicate and repeated at least three times. For Oil red O staining, the medium was removed, and the cells were washed with ddH2O and fixed with 2% formalin overnight. Oil red dye was added after washing and incubated for 1 h at room temperature. After washing, pictures were taken under the microscope. For mitochondrial MitoTracker staining, the medium from the cells was removed and replaced with prewarmed medium with MitoTracker dye, followed by incubation for 30 min at 37°C in a 5% CO2 atmosphere. After washing with prewarmed PBS, the fluorescence was observed under green excitation light. For MitoSOX staining, primary hepatocytes were washed three times with 1× PBS, followed by incubation with MitoSOX Red dye for 30 min at 37°C in the dark. Hepatocytes were then washed three times with 1× PBS, and fluorescence was examined under a fluorescence microscope. For trypan blue staining, primary hepatocytes were washed followed by incubation with trypan blue solution at room temperature. Hepatocytes were then washed three times with 1× PBS and photographed by microscopy. For C11-bodipy staining, cells were incubated with C11-bodipy dye at a final concentration of 5 µM for 30 min at 37°C in the dark. Then, the washed cells were viewed and photographed by microscopy. ImageJ software was used to quantify cell diameter and the relative density of the Oil red O, MitoTracker, MitoSOX, C11-bodipy and Grx1-roGFP2/roGFP2-Orp1 probe staining results.

Oxygen consumption rates of primary hepatocytes

The cell oxygen consumption rate (OCR) was measured using an oxygen meter (Strathkelvin Instruments) with a Mitocell (MT200) mixing chamber. The primary hepatocytes were scraped off with a cell scraper and suspended in 400 µl cell culture medium to measure the OCR of the cells. The OCR was calculated using software (782 Oxygen System version 4.0).

Gene expression analyses

Mouse livers were extracted and immediately frozen in liquid nitrogen. Total RNA from tissue or hepatocytes was extracted using the TRIzol method following the manufacturer’s instructions. For RT-qPCR, 2 µg of RNA was reverse-transcribed using HiScript II Q RT SuperMix (Vazyme, R222-01) followed by qPCR using SYBR Green (Bimake, B21203). Relative mRNA expression was normalized to the expression of AP0. The primers used for gene expression are listed in Table S3.

Immunoblotting analysis

Mouse hepatocyte lysates were prepared in lysis buffer containing 50 mM Tris-HCl (pH = 7.8), 137 mM NaCl, 10 mM NaF, 1 mM EDTA, 1% Triton X-100, 10% glycerol, and a protease inhibitor cocktail (Bimake, B14002) after three freeze/thaw cycles. The antibodies used were HSP90 (13171-1-AP) from Proteintech; phospho-AMPK (2535S), AMPK (2532), and phospho-AKT (4060 T) from Cell Signaling Technology; total OXPHOS (ab110413) from Abcam; Slc7a11 (PA1-16893) from Invitrogen; and AKT (10176-2-AP) from Proteintech.

GSH and GSSG assay

GSH levels were measured using a GSH and GSSG Assay kit (Beyotime, S0053) according to the manufacturer’s instructions. The primary hepatocytes were centrifuged after washing. The protein removal reagent M solution was added to the cell precipitate at a three times volume, and the sample was vortexed fully. Then, the sample was quickly frozen and thawed twice in liquid nitrogen and a 37°C water bath, followed by centrifugation at 4°C for 10,000 × g for 10 min. The supernatant was separated into two groups and diluted ten times for the detection of total GSH and GSSG content. One group was treated with GSH clearing solution and incubated at room temperature for 1 h to detect GSSG content. The other group was directly used for the detection of total GSH. After adding the total GSH detection working solution, the sample was incubated for 5 min, and then 0.5 mg/ml NADPH was added to determine A412. To calculate the GSH content, first, the absorbance value at different time points was obtained to calculate ΔA412/min. Then, the concentration of the standard and corresponding ΔA412/min value were used to draw a standard curve. The content of total GSH or GSSG in the sample was calculated according to the ΔA412/min value of the sample by referring to the standard curve. The content of reduced GSH was calculated by subtracting the content of GSSG from the content of total GSH (reduced GSH = Total GSH-GSSG × 2). The total GSH, GSSG and reduced GSH contents were normalized by the protein concentration of the cells.

RNA-seq data analysis

The fastq files were subjected to quality control using FastQC, followed by alignment against the mouse reference genome (mm10) using the aligner HISAT2 [37]. HTSeq was used to count reads mapped to each reference gene and U12 intron location [38]. DESeq2 was used for differential expression analysis [39]. Gene enrichment analysis was performed using DAVID (https://david.ncifcrf.gov/).

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9. Statistical differences were evaluated using two-tailed unpaired Student’s t test for comparisons between two groups or analysis of variance (ANOVA) and appropriate post hoc analyses for comparisons of more than two groups. A p value of less than 0.05 (*p < 0.05, **p < 0.01, and ***p < 0.001) was considered statistically significant. Statistical methods and corresponding p values for data shown in each panel are included in the figure legends.

Results

Fatty acid metabolism is the most significantly reduced metabolic process after primary hepatocyte isolation and culture

To explore the metabolic reprograming of primary hepatocytes after isolation and culture, we extracted primary hepatocytes from mice using a two-step collagenase perfusion method and performed RNA sequencing using isolated hepatocytes cultured in vitro for 0, 24 and 72 h. We noticed that a large number of differentially expressed genes (DEGs) were progressively upregulated or downregulated after 24 h and 72 h of in vitro culture of hepatocytes (Figure 1A and Table S1). Then, we subjected upregulated and downregulated DEGs at 72 h after in vitro culture to GO biological pathway and cellular component enrichment analysis. We found that the downregulated DEGs were largely clustered to lipid metabolism and fatty acid metabolic processes, while the upregulated DEGs were mainly associated with cytoplasmic translation in biological process enrichment analyses. The cellular component analysis indicated that the downregulated DEGs were mainly mitochondrial component-associated genes (Figure 1B and Fig S1). Gene set enrichment analysis (GSEA) indicated that the fatty acid metabolism pathway was drastically compromised (Figure 1C). The Molecular Complex Detection (MCODE) network showed that the downregulated DEGs constituted 12 densely connected networks. Among them, monocarboxylic acid metabolic process, fatty acid oxidation, and the SREBP signaling pathway, which regulates fatty acid synthesis and oxidation, were heavily affected. Notably, cysteine and methionine metabolism and glutathione metabolism, which are critical for redox homoeostasis, and the complement cascade pathway were also reduced in cultured primary hepatocytes (Figure 1D). Significant enrichment of the transcription factors Srebf1 and Pparα was observed in transcription factor clustering analyses (TRRUST), which are key regulators of de novo lipogenesis and fatty acid oxidation (Figure 1E).

Figure 1.

Figure 1.

Open in a new tab

Fatty acid metabolism in primary hepatocytes is the most significantly compromised metabolic process after isolation and culture. A. Heatmap showing the differential genes of RNA sequencing of primary hepatocytes after isolation for 0, 24 and 72 h. B. Enrichment analyses of genes downregulated in primary hepatocytes at 72 h after isolation and culture. C. GSEA shows the enrichment of fatty acid metabolic processes in primary hepatocytes at 72 h after isolation and culture. D. MCODE enrichment of genes in primary hepatocytes at 72 h after isolation and culture. E. TRRUST analysis shows the enriched transcription factors in primary hepatocytes at 72 h after isolation and culture.

Consistent with the results of RNA-seq, qPCR analysis showed that the expression of fatty acid synthesis-related genes (Srebp1c, Fasn, Scd1, Fsp27) and fatty acid oxidation-related genes (Pparα, Fgf21) was constantly downregulated during the isolation and culture of primary hepatocytes within three days (Figure 2A). We also observed that the typical hepatocyte morphology, lipid content and mitochondrial mass of cultured primary hepatocytes were continuously lost within three days of primary culture of hepatocytes (Figure 2B). Notably, mitochondrial respiration function gradually decreased in the isolated primary hepatocytes along with the loss of mitochondrial mass (Figure 2C). All these results indicate that the ability of cultured primary hepatocytes to mediate fatty acid metabolism was gradually lost after isolation and culture.

Figure 2.

Figure 2.

Open in a new tab

Fatty acid metabolic gene expression, morphology, lipid content, mitochondrial mass and respiration function of primary hepatocytes gradually decreased after isolation and culture. A. qPCR analyses of fatty acid synthesis- and oxidation-related gene expression in the liver (L) and primary hepatocytes after isolation for the indicated times. B. DIC morphology, Oil red O (ORO) and MitoTracker staining of primary hepatocytes after isolation and culture for the indicated times. Scale bar = 100µm. The quantification results are shown on the right. C. Oxygen consumption rates of primary hepatocytes treated with vehicle (veh), oligomycin (Oligo, 20 µg/ml) and Fccp (15 µM) after isolation and culture for the indicated times. Data represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired Student’s t test.

GSH content is reduced, leading to spontaneous ferroptosis after primary hepatocyte isolation and culture

By analyzing the RNA sequencing results, we noticed a significant decrease in glutathione metabolic processes along with the suppression of fatty acid metabolic processes after primary hepatocyte isolation and culture. Considering that the cellular redox environment plays a vital role in the maintenance of cellular homeostasis and metabolic functions, we measured intracellular GSH levels in the liver and primary hepatocytes after isolation and culture for 1, 2 and 3 days with or without dexamethasone (dex) treatment, one of the most commonly used hormone-based additives, which regulates fatty acid metabolism via the activation of liver-specific transcriptional programs by enhancing the expression of key hepatic transcription factors and increasing their DNA-binding activity. We found that intracellular GSH content, including total glutathione, oxidized glutathione and reduced glutathione, was significantly downregulated after isolation and culture of primary hepatocytes. Dex treatment partially reversed the downregulation of GSH contents (Figure 3A). A similar trend was shown in the expression of the GSH synthesis-related genes Nrf2, Gclc, Gclm, Gss and Ggt (Figure 3B). Interestingly, we found that the expression of Slc7a11, a key subunit of the Xc glutamate/cystine reverse transporter, was significantly upregulated after one day of isolation and culture of primary hepatocytes (Figure 3C). To further verify the change in GSH and ROS content in primary hepatocytes after isolation and culture, we expressed the GSH fluorescent probe Grx1-roGFP2 and the ROS fluorescent probe Orp1-roGFP2 in the liver via adeno-associated virus (AAV) infection under a liver-specific TBG promoter. After isolation and culture of hepatocytes expressing these roGFP2 probes, we revealed that fluorescence from Grx1 and Orp1 roGFP2 probes, which indicates GSH and ROS content in primary hepatocytes, was drastically decreased. Additionally, MitoSOX staining indicated that mitochondrial ROS content was also decreased in primary hepatocytes (Figure 3D). Interestingly, C11-bodipy staining indicated that hepatocyte lipid peroxidation and trypan blue-stained dead cells were increased, unveiling that some isolated hepatocytes undergo spontaneous ferroptosis under culture condition, which is negatively associated with a decrease in intracellular GSH content (Figure 3E). These results indicate that the dramatic loss of GSH and ROS content may disrupt redox homeostasis, while upregulated lipid peroxidation due to reduced GSH levels drives ferroptosis after primary culture of hepatocytes, thus compromising fatty acid metabolism in primary hepatocytes.

Figure 3.

Figure 3.

Open in a new tab

Redox activity in primary hepatocytes is attenuated, leading to spontaneous ferroptosis after isolation and culture. A. Total GSH, GSSG and reduced GSH content in primary hepatocytes with or without dexamethasone (Dex, 0.5 µM) treatment after isolation and culture at the indicated times. B-C. GSH synthesis-related gene (B) and Slc7a11 (C) expression in the liver and primary hepatocytes with or without dexamethasone (Dex, 0.5 µM) treatment after isolation and culture for the indicated times. D. Fluorescence images of primary hepatocytes isolated from mice injected with Grx1- and Orp1-roGFP2-expressing AAV virus and then cultured for the indicated times and MitoSOX staining of primary hepatocytes cultured for the indicated times. Scale bar = 100µm. The quantification results are shown on the right. E. Images of trypan blue staining and fluorescence images of C11-bodipy staining of primary hepatocytes isolated from mice and then cultured for the indicated times. Scale bar = 100µm. The quantification results are shown on the right. Data represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired Student’s t test.

Expression of slc7a11 regulates GSH production and fatty acid metabolism in primary hepatocytes

The Xc-glutamate/cystine reverse transporter is a crucial mitochondrial intermembrane transporter that imports cystine and exports glutamate. This process is critical for GSH synthesis. To explore the relationship between elevated Slc7a11 expression and devitalized fatty acid metabolism after isolation and culture of primary hepatocytes, we isolated primary hepatocytes from the liver overexpressing Slc7a11 via AAV virus infection under a liver-specific TBG promoter and GFP-expressing control (Figure 4A). In addition, we supplemented the medium with dex. We found that overexpression of Slc7a11 in the liver further elevated dex-increased expression of Nrf2, a master regulator of GSH synthesis, and GSH content (Figure 4B-C), while Slc7a11 overexpression in hepatocytes also increased the expression of the fatty acid synthesis-related genes Srebp1c, Fasn, Scd1, and Fsp27 and the fatty acid oxidation-related genes Pparα and Fgf21 upon dex treatment (Figure 4D). Subsequently, we administered AAV virus expressing cre recombinase under the TBG promoter to Slc7a11 flox/flox mice, thereby specifically knocking out Slc7a11 in the liver (Figure 5A and Fig S2). We observed reduced GSH content in primary hepatocytes after depletion of Slc7a11, although GSH synthesis-related genes were not altered (Figure 5B-C). The genes that regulate fatty acid metabolism were significantly decreased in Slc7a11-deficient hepatocytes, and dex treatment was not sufficient to overcome this defect (Figure 5D). This is consistent with the results of overexpression of Slc7a11.

Figure 4.

Figure 4.

Open in a new tab

Overexpression of Slc7a11 in the liver before primary hepatocyte isolation increases GSH content and facilitates the preservation of fatty acid metabolism upon dexamethasone treatment in isolated primary hepatocytes. A-B. Protein and mRNA expression of Slc7a11 (A) and mRNA expression of GSH synthesis-related genes (B) in primary hepatocytes treated with Veh or dexamethasone (Dex, 0.5 µM) for 72 h after isolation. C. Total GSH, GSSG and reduced GSH content of Slc7a11-expressing primary hepatocytes treated with Veh or Dex (0.5 µM) for 72 h after isolation. D. qPCR analyses of fatty acid metabolism-related genes in Slc7a11-expressing primary hepatocytes treated with Veh or Dex (0.5 µM) for 72 h after isolation. Data represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired Student’s t test.

Figure 5.

Figure 5.

Open in a new tab

Hepatic depletion of Slc7a11 decreases GSH content and accelerates the loss of fatty acid metabolism activity in isolated primary hepatocytes. A-B. Protein and mRNA expression of Slc7a11 (A) and mRNA expression of GSH synthesis-related genes (B) in primary hepatocytes isolated from Slc7a11 flox/flox mice injected with AAV-TBG-cre virus and treated with Veh or Dex (0.5 µM) for 72 h after isolation. C. Total GSH, GSSG and reduced GSH content of Slc7a11 knockout primary hepatocytes treated with Veh or Dex (0.5 µM) for 72 h after isolation. D. qPCR analyses of fatty acid metabolism-related genes in Slc7a11 knockout primary hepatocytes treated with Veh or Dex (0.5 µM) for 72 h after isolation. Data represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired Student’s t test.

Expression of Slc7a11 synergizes with dex treatment to reserve fatty acid metabolism in primary hepatocytes

To globally analyze the potential synergistic effect of dex treatment and Slc7a11 expression in maintaining fatty acid metabolism in primary hepatocytes, we performed RNA sequencing of Slc7a11-overexpressing primary hepatocytes at 72 h after isolation with or without dex treatment. Our data showed that overexpression of Slc7a11 largely amplified the expression of a group of dex-induced genes (Figure 6A and Table S2). The enrichment analyses indicated that these Slc7a11 and dex synergistically upregulated genes are involved in lipid metabolic processes (Figure 6B). To assess whether primary hepatocytes expressing Slc7a11 facilitate lipogenesis and fatty acid oxidation upon hormone-induced activation, we treated Slc7a11-expressing primary hepatocytes with T0901317, an agonist of LXR that induces de novo lipogenesis, and GW7647, a ligand for Pparα, which increases hepatic β-oxidation. Upon treatment with T0901317, the expression of Fsp27, Fasn, Srebp1c, and Scd1 was upregulated, and overexpressing Slc7a11 drove a more profound elevation of lipogenic genes, while GW7647 treatment further induced Fgf21 expression in Slc7a11-overexpressing cells compared with control cells. In contrast, knockout of Slc7a11 in primary hepatocytes suppressed lipogenic gene and fatty acid β-oxidation gene expression induced by T0901317 and GW7647 treatment (Figure 6C-D), indicating that Slc7a11 expression in primary hepatocytes amplifies hormone-induced primary hepatocyte fatty acid metabolic activities. These data suggest that Slc7a11 expression together with dex treatment preserves basal and hormone-induced fatty acid metabolism.

Figure 6.

Figure 6.

Open in a new tab

Hepatic Slc7a11 expression and dex treatment synergistically preserve fatty acid metabolism in isolated primary hepatocytes. A. Heatmap showing the differentially expressed genes identified by RNA sequencing of vector (Vec)- and Slc7a11-expressing primary hepatocytes treated with Veh or Dex (0.5 µM). B. Enrichment analysis of genes synergistically induced by Slc7a11 and dex treatment. C-D. qPCR analyses of fatty acid metabolism-related genes in Slc7a11-overexpressing (C) and Slc7a11-knockout (D) primary hepatocytes treated with Veh or T0901317 (5 µM) and GW7647 (1 µM) for 72 h after isolation. Data represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired Student’s t test.

Slc7a11 regulates AKT and AMPK signaling and mitochondrial homeostasis in primary hepatocytes

Insulin and AMPK signaling are critical for fatty acid metabolism, and we then examined the activation of AKT, a downstream factor in insulin signaling, and AMPK pathways at 0, 24 and 72 h in primary hepatocytes isolated in culture. We observed that the phosphorylation and total levels of AKT and AMPK were downregulated over time after isolation and culture of primary hepatocytes. Notably, dex treatment slightly alleviated the downregulation of the expression of these factors in primary hepatocytes after 72 h of culture (Figure 7A and Fig S3). The overexpression of Slc7a11 in isolated primary hepatocytes synergistically protected against the loss of AKT phosphorylation with dex treatment, while knockout of Slc7a11 further decreased the expression of phosphorylated AKT. Although Slc7a11 overexpression had no effect on the expression of phosphorylated AMPK, Slc7a11 knockout attenuated AMPK phosphorylation (Figure 7B-C and Fig S3). These results indicate that Slc7a11 expression may preserve fatty acid metabolism in primary hepatocytes by regulating the AKT and AMPK signaling pathways.

Figure 7.

Figure 7.

Open in a new tab

Expression of Slc7a11 regulates AKT and AMPK signaling and mitochondrial respiration complex expression in isolated primary hepatocytes. A-C. Western blot analysis of the phosphorylation and total levels of AKT and AMPK and Slc7a11 expression in primary hepatocytes isolated at the indicated times (A) or in Slc7a11-expressing (B) and Slc7a11-knockout (C) primary hepatocytes treated with Veh or Dex (0.5 µM) for 72 h. D-E. Western blot analysis of mitochondrial respiration complex expression in primary hepatocytes isolated at the indicated times (D) or in Slc7a11-expressing (E) and Slc7a11 knockout (F) primary hepatocytes treated with Veh or Dex (0.5 µM) for 72 h. Data are representative of three independent experiments.

Mitochondrial homeostasis greatly influences fatty acid oxidation, which produces acetyl-CoA for respiration, de novo lipogenesis and cellular redox homeostasis. Our previous RNA sequencing results suggest that the expression and function of components of the mitochondrial respiratory chain complex are downregulated after hepatocyte isolation and culture. MitoTracker and MitoSOX staining also supported that mitochondrial mass and ROS production were decreased in isolated primary hepatocytes. By measuring the expression of a component protein in each respiratory chain complex, we revealed that compared to freshly isolated primary hepatocytes, ATP5A, UQCRC2, MTCO1 and NDUFB8 expression was gradually suppressed in primary hepatocytes after in vitro culture, which may limit fatty acid oxidation activities in the cell (Figure 7D and Fig S3). Then, we overexpressed and knocked out Slc7a11 in isolated hepatocytes to explore whether Slc7a11 regulates GSH content and plays a role in maintaining mitochondrial homeostasis. The results showed that Slc7a11 overexpression upregulated MTCO1 and SDHB expression, while Slc7a11 deletion decreased MTCO1, SDHB and NDUFB8 expression (Figure 7E-F and Fig S3). These results imply that Slc7a11 expression controls mitochondrial respiratory chain complex expression, which may contribute to mitochondrial homeostasis and fatty acid oxidation in mitochondria.

Supplementation with GSH and suppression of ferroptosis preserves fatty acid metabolism in primary hepatocytes

Slc7a11 mediates redox homeostasis and resists ferroptosis by generating GSH. To verify that GSH produced by Slc7a11 is a key factor that counteracts ferroptosis and maintains fatty acid metabolism in isolated primary hepatocytes, we supplemented reduced GSH ethyl ester (GSH-MEE) in the culture medium of primary hepatocytes immediately after isolation. We found that GSH-MEE supplementation decreased Slc7a11 expression but had no effect on GSH synthesis-related genes, while intracellular GSH levels were increased (Figure 8A-B). We also observed elevated expression of genes related to fatty acid synthesis and oxidation after GSH-MEE supplementation, and this GSH-mediated upregulation was synergistic with dex treatment (Figure 8C), indicating that increasing GSH content in primary hepatocytes is sufficient to promote fatty acid metabolism in cells. We also examined the phosphorylation of AKT and AMPK. GSH-MEE supplementation increased the level of phosphorylated AMPK but not AKT, which is partially consistent with the results of Slc7a11 overexpression (Figure 8D and Fig S4). In addition, we also assessed mitochondrial respiratory chain complex expression. The results revealed that GSH-MEE treatment restored the decrease in ATP5A, UQCRC2, MTCO1, SDHB and NDUFB8 expression, suggesting that GSH also plays a role in maintaining mitochondrial activities in primary hepatocytes (Figure 8E and Fig S4). Furthermore, we treated freshly isolated primary hepatocytes with ferrostatin-1 (Fer-1), a selective ferroptosis inhibitor. Slc7a11 expression was induced after treatment, along with increased fatty acid metabolic gene expression, which was synergistic with dex treatment (Figure 8F). In addition, suppression of ferroptosis increased both AKT and AMPK phosphorylation (Figure 8G and Fig S4). Importantly, Fer-1 treatment slightly decreased UQCRC2, MTCO1 and NDUFB8 expression, indicating that the protective effect of GSH against mitochondrial loss is not due to reducing ferroptosis (Figure 8H and Fig S4). Together, our data suggest that the preservation of fatty acid metabolism in isolated primary hepatocytes by Slc7a11 expression may occur through an increase in cellular GSH content, thereby reducing spontaneous ferroptosis. Supplementation with GSH-MEE or inhibition of ferroptosis largely improves the fatty acid metabolism capacity of primary hepatocytes.

Figure 8.

Figure 8.

Open in a new tab

GSH supplementation and ferroptosis inhibition in isolated hepatocytes preserves fatty acid metabolism, AKT and AMPK signaling and mitochondrial respiration complex expression. A. qPCR analyses of GSH synthesis-related genes treated with Veh and Dex (0.5 µM) and reduced GSH ethyl ester (GSH-MEE, 0.5 mM) in combination for 72 h after isolation. B. Total GSH, GSSG and reduced GSH content of primary hepatocytes with Veh and GSH-MEE (0.5 mM) in combination for 72 h after isolation. C. qPCR analyses of fatty acid metabolism-related genes treated with Veh and Dex (0.5 µM) and GSH-MEE (0.5 mM) in combination for 72 h after isolation. D-E. Western blot analyses of phosphorylation and total levels of AKT and AMPK and Slc7a11 expression and mitochondrial respiration complex expression in primary hepatocytes treated with Veh and Dex (0.5 µM) and GSH-MEE (0.5 mM) in combination for 72 h after isolation. F. qPCR analyses of fatty acid metabolism-related genes treated with Veh and Dex (0.5 µM) and ferrostatin-1 (Fer-1, 0.5 µM) in combination for 72 h after isolation. G-H. Western blot analyses of phosphorylation and total levels of AKT and AMPK and Slc7a11 expression and mitochondrial respiration complex expression in primary hepatocytes treated with Veh and Dex (0.5 µM) and Fer-1 (0.5 µM) in combination for 72 h after isolation. Data represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired Student’s t test. Data in D-E and G-H are representative of three independent experiments.

Discussion

Primary hepatocytes have been extensively used in distinct scenarios for studying liver biology and largely applied to molecular research exploring liver metabolic functions. However, during the isolation and culture of hepatocytes, many metabolic processes are reprogramed, and their activities are decreased. As such, the discovery of a potential approach that is able to preserve hepatocyte metabolic activities after isolation and culture is essential. Here, we focus on illustrating which metabolic pathway is the most affected in in vitro culture and subsequent functional analyses using primary hepatocytes and aim to demonstrate a key underlying mechanism of this dysregulation, which could be beneficial for future in vitro studies of the liver. In this study, we first performed a comprehensive RNA sequencing analysis using isolated hepatocytes at a close time course after isolation. We clarified that fatty acid metabolism is the most significant loss-of-function metabolic process in isolated primary hepatocytes. Interestingly, the disruption of redox homeostasis tightly correlated with the drastic drop in fatty acid metabolic activity in cultured hepatocytes, implying that a change in redox status in hepatocytes could be a potential mechanism that influences metabolic activities in primary hepatocytes. Notably, Slc7a11, a cystine/glutamate antiporter that is closely associated with GSH synthesis, was highly induced after isolation of primary hepatocytes in response to the decrease in GSH content. Thus, we overexpressed and knocked out Slc7a11 in the liver before hepatocyte isolation and monitored GSH levels, fatty acid synthesis and oxidation gene expression and their regulatory signaling immediately after isolation. Our results indicate that Slc7a11 expression induces fatty acid metabolic activity, especially under dex treatment. The RNA sequencing data showed that the expression of a set of genes was upregulated in hepatocytes expressing Slc7a11 together with dex treatment, and these genes are implicated in the fatty acid metabolic process. Intriguingly, Slc7a11-expressing primary hepatocytes have higher sensitivity to hormone-induced fatty acid metabolic events, such as T0901317-induced de novo lipogenesis and GW7647-induced fatty acid β-oxidation, indicating that Slc7a11 expression is crucial for primary hepatocyte metabolic functions. In addition, Slc7a11 expression also increased AKT and AMPK phosphorylation, which are upstream pathways that govern fatty acid synthesis and oxidation in hepatocytes. Surprisingly, Slc7a11 expression also affects mitochondrial respiration chain complex expression, which is decreased in primary hepatocytes after isolation. Eventually, we verified that GSH levels in primary hepatocytes directly dominate Slc7a11 expression-induced beneficial effects in primary hepatocytes, including preservation of fatty acid metabolic activity and mitochondrial function. GSH is an antioxidant and increases the reducing capacity of cells. Decreased GSH levels are highly associated with an increase in ferroptosis, a new type of programed cell death induced by iron-dependent lipid peroxidation. Notably, isolated primary hepatocytes undergo ferroptosis in in vitro culture, which contributes to the dysfunction of fatty acid metabolism in these ferroptotic primary hepatocytes. In addition, the reducing environment with a high GSH amount is critical for the prevention of oxidative stress, which may be harmful for metabolic functions in isolated hepatocytes, although we indeed observed a decrease in ROS levels in isolated primary hepatocytes by the roGFP2-Orp1 probe and MitoSOX staining. The detailed mechanisms that drive ferroptosis and the disorder of redox homeostasis and their roles in the loss of hepatic fatty acid metabolic activities still need to be further explored.

Conclusions

Overall, in this study, we demonstrated that hepatic Slc7a11 expression is sufficient to preserve fatty acid metabolic activities in synergy with dex treatment by increasing GSH levels and reducing ferroptosis, thus providing a promising approach that better reduces the dysfunctional reprograming of hepatocytes in culture for more precise biological research using primary hepatocytes.

Acknowledgments

We thank the members of the Zhao lab for their delicate guidance and advice on this study.

Funding Statement

This work was funded by the National Key R&D Program of China (2020YFA0803603 to X.Y.Z), the National Natural Science Foundation of China (82070894 to X.Y. Z), Science and Technology Commission of Shanghai Municipality (21ZR1436500, 22ZR1479800 to X.Y.Z), Innovative research team of high-level local universities in Shanghai (SHSMU-ZDCX20212501 to X.Y.Z), Shanghai Frontiers Science Center of Cellular Homeostasis and Human Diseases.

Author contributions

Y.L., Y.F., K.W., W.L. and X.Y.Z. conceived the project and designed the research. Y.L., K.W. and Y.F. performed the studies. Y.L. W.L. and X.Y.Z. analyzed the data and wrote the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The authors confirm that the datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

References

  • 1.Ogoke O, Oluwole J, Parashurama N.. Bioengineering considerations in liver regenerative medicine. J Biol Eng. 2017;11:46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Gomez-Lechon MJ, Donato MT, Castell JV, et al. Human hepatocytes as a tool for studying toxicity and drug metabolism. Curr Drug Metab. 2003 Aug;4(4):292–312. [DOI] [PubMed] [Google Scholar]
  • 3.Wilkening S, Stahl F, Bader A.. Comparison of primary human hepatocytes and hepatoma cell line Hepg2 with regard to their biotransformation properties. Drug Metab Dispos. 2003 Aug;31(8):1035–1042. [DOI] [PubMed] [Google Scholar]
  • 4.Andersson TB, Kanebratt KP, Kenna JG.. The HepaRG cell line: a unique in vitro tool for understanding drug metabolism and toxicology in human. Expert Opin Drug Metab Toxicol. 2012 Jul;8(7):909–920. [DOI] [PubMed] [Google Scholar]
  • 5.Samanez CH, Caron S, Briand O, et al. The human hepatocyte cell lines IHH and HepaRG: models to study glucose, lipid and lipoprotein metabolism. Arch Physiol Biochem. 2012 Jul;118(3):102–111. [DOI] [PubMed] [Google Scholar]
  • 6.Gerbal-Chaloin S, Funakoshi N, Caillaud A, et al. Human induced pluripotent stem cells in hepatology: beyond the proof of concept. Am J Pathol. 2014 Feb;184(2):332–347. [DOI] [PubMed] [Google Scholar]
  • 7.Bell CC, Lauschke VM, Vorrink SU, et al. Transcriptional, functional, and mechanistic comparisons of stem cell-derived hepatocytes, HepaRG cells, and three-dimensional human hepatocyte spheroids as predictive In vitro systems for drug-induced liver injury. Drug Metab Dispos. 2017 Apr;45(4):419–429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Godoy P, Hewitt NJ, Albrecht U, et al. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol. 2013 Aug;87(8):1315–1530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Fraczek J, Bolleyn J, Vanhaecke T, et al. Primary hepatocyte cultures for pharmaco-toxicological studies: at the busy crossroad of various anti-dedifferentiation strategies. Arch Toxicol. 2013 Apr;87(4):577–610. [DOI] [PubMed] [Google Scholar]
  • 10.Papeleu P, Loyer P, Vanhaecke T, et al. Proliferation of epidermal growth factor-stimulated hepatocytes in a hormonally defined serum-free medium. Altern Lab Anim. 2004 Jun;32(Suppl 1A):57–64. [DOI] [PubMed] [Google Scholar]
  • 11.Elaut G, Henkens T, Papeleu P, et al. Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures. Curr Drug Metab. 2006 Aug;7(6):629–660. [DOI] [PubMed] [Google Scholar]
  • 12.Baker TK, Carfagna MA, Gao H, et al. Temporal gene expression analysis of monolayer cultured rat hepatocytes. Chem Res Toxicol. 2001 Sep;14(9):1218–1231. [DOI] [PubMed] [Google Scholar]
  • 13.de Longueville F, Atienzar FA, Marcq L, et al. Use of a low-density microarray for studying gene expression patterns induced by hepatotoxicants on primary cultures of rat hepatocytes. Toxicol Sci. 2003 Oct;75(2):378–392. [DOI] [PubMed] [Google Scholar]
  • 14.Rodriguez-Antona C, Donato MT, Boobis A, et al. Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells. Xenobiotica. 2002 Jun;32(6):505–520. [DOI] [PubMed] [Google Scholar]
  • 15.Beigel J, Fella K, Kramer PJ, et al. Genomics and proteomics analysis of cultured primary rat hepatocytes. Toxicol Vitro. 2008 Feb;22(1):171–181. [DOI] [PubMed] [Google Scholar]
  • 16.Heslop JA, Rowe C, Walsh J, et al. Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile. Arch Toxicol. 2017 Jan;91(1):439–452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Matsuno F, Chowdhury S, Gotoh T, et al. Induction of the C/EBP beta gene by dexamethasone and glucagon in primary-cultured rat hepatocytes. J Biochem. 1996 Mar;119(3):524–532. [DOI] [PubMed] [Google Scholar]
  • 18.Yamada S, Otto PS, Kennedy DL, et al. The effects of dexamethasone on metabolic activity of hepatocytes in primary monolayer culture. In Vitro. 1980 Jul;16(7):559–570. [DOI] [PubMed] [Google Scholar]
  • 19.Bell CC, Dankers ACA, Lauschke VM, et al. Comparison of hepatic 2D sandwich cultures and 3D spheroids for long-term toxicity applications: a multicenter study. Toxicol Sci. 2018 Apr 1;162(2):655–666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Lauschke VM, Shafagh RZ, Hendriks DFG, et al. 3D primary hepatocyte culture systems for analyses of liver diseases, drug metabolism, and toxicity: emerging culture paradigms and applications. Biotechnol J. 2019 Jul;14(7):e1800347. [DOI] [PubMed] [Google Scholar]
  • 21.Bannai S. Exchange of cystine and glutamate across plasma membrane of human fibroblasts. J Biol Chem. 1986 Feb 15;261(5):2256–2263. [PubMed] [Google Scholar]
  • 22.Sato H, Tamba M, Ishii T, et al. Cloning and expression of a plasma membrane cystine/glutamate exchange transporter composed of two distinct proteins. J Biol Chem. 1999 Apr 23;274(17):11455–8. [DOI] [PubMed] [Google Scholar]
  • 23.Nakamura E, Sato M, Yang H, et al. 4F2 (CD98) heavy chain is associated covalently with an amino acid transporter and controls intracellular trafficking and membrane topology of 4F2 heterodimer. J Biol Chem. 1999 Jan 29;274(5):3009–3016. [DOI] [PubMed] [Google Scholar]
  • 24.Koppula P, Zhang Y, Zhuang L, et al. Amino acid transporter SLC7A11/xCT at the crossroads of regulating redox homeostasis and nutrient dependency of cancer. Cancer Commun (Lond). 2018 Apr 25;38(1):12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Lewerenz J, Hewett SJ, Huang Y, et al. The cystine/glutamate antiporter system x(c)(-) in health and disease: from molecular mechanisms to novel therapeutic opportunities. Antioxid Redox Signal. 2013 Feb 10;18(5):522–555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Koppula P, Zhuang L, Gan B.. Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy. Protein Cell. 2021 Aug;12(8):599–620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Forman HJ, Zhang H, Rinna A.. Glutathione: overview of its protective roles, measurement, and biosynthesis. Mol Aspects Med. 2009 Feb-Apr;30(1-2):1–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Vaskova J, Kocan L, Vasko L, et al. Glutathione-Related enzymes and proteins: A review. Molecules. 2023 Feb 2;28(3):1447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Chen Y, Dong H, Thompson DC, et al. Glutathione defense mechanism in liver injury: insights from animal models. Food Chem Toxicol. 2013 Oct;60:38–44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Liu T, Sun L, Zhang Y, et al. Imbalanced GSH/ROS and sequential cell death. J Biochem Mol Toxicol. 2022 Jan;36(1):e22942. [DOI] [PubMed] [Google Scholar]
  • 31.Yuan L, Kaplowitz N.. Glutathione in liver diseases and hepatotoxicity. Mol Aspects Med. 2009 Feb-Apr;30(1-2):29–41. [DOI] [PubMed] [Google Scholar]
  • 32.Saito C, Zwingmann C, Jaeschke H.. Novel mechanisms of protection against Acetaminophen hepatotoxicity in mice by glutathione and N-acetylcysteine. Hepatology. 2010 Jan;51(1):246–254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Irie M, Sohda T, Anan A, et al. Reduced glutathione suppresses oxidative stress in nonalcoholic fatty liver disease. Euroasian J Hepatogastroenterol. 2016 Jan-Jun;6(1):13–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Hajighasem A, Farzanegi P, Mazaheri Z.. Effects of combined therapy with resveratrol, continuous and interval exercises on apoptosis, oxidative stress, and inflammatory biomarkers in the liver of old rats with non-alcoholic fatty liver disease. Arch Physiol Biochem. 2019 May;125(2):142–149. [DOI] [PubMed] [Google Scholar]
  • 35.Ursini F, Maiorino M.. Lipid peroxidation and ferroptosis: the role of GSH and GPx4. Free Radic Biol Med. 2020 May 20;152:175–185. [DOI] [PubMed] [Google Scholar]
  • 36.Lin J, Wu PH, Tarr PT, et al. Defects in adaptive energy metabolism with CNS-linked hyperactivity in PGC-1alpha null mice. Cell. 2004 Oct 1;119(1):121–135. [DOI] [PubMed] [Google Scholar]
  • 37.Dobin A, Davis CA, Schlesinger F, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013 Jan 1;29(1):15–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Anders S, Pyl PT, Huber W.. HTSeq–a python framework to work with high-throughput sequencing data. Bioinformatics. 2015 Jan 15;31(2):166–169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Love MI, Huber W, Anders S.. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The authors confirm that the datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Articles from Redox Report : Communications in Free Radical Research are provided here courtesy of Taylor & Francis

RESOURCES