hepg2 hepatoma cell line  (ATCC)

Journal: International Journal of Molecular Sciences

Article Title: Gut Microbiota-Derived Propionic Acid Mediates ApoA-I-Induced Amelioration of MASLD via Activation of GPR43–Ca 2+ –CAMKII–ATGL Hepatic Lipolysis

doi: 10.3390/ijms27010468

Figure Lengend Snippet: PPA-mediated reduction in lipid accumulation involves enhanced lipolysis and fatty acid oxidation in HepG2 cells. ( A ) Following 24 h incubation with 500 μM oleic acid (OA) or bovine serum albumin (BSA), HepG2 cells were exposed to PPA at concentrations of 0, 0.5, 0.75, 1, or 5 mM for an additional 48 h. Cell viability was determined via the CCK-8 assay. ( B ) Oil Red O staining images illustrate lipid droplets (scale bar = 200 μm). ( C ) Quantitative assessment of lipid content from Oil Red O staining. ( D ) Fluorescence images of BODIPY 493/503-stained lipid droplets (scale bar = 25 μm). ( E ) Mean fluorescence intensity quantification of BODIPY 493/503 staining. ( F , G ) Western blot analysis for detecting phosphorylated ATGL (p-ATGL) and total ATGL (t-ATGL). ( H , I ) CPT1A expression levels were examined by Western blotting. Colors represent the following groups: control (gray), OA alone (green), OA plus PPA at the indicated concentrations (orange). Group labels are displayed on the x-axis. Data represent mean ± SEM; n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns represents no significant difference.

Article Snippet: The HepG2 human hepatoma cell line (obtained from ATCC) was maintained under standard conditions at 37 °C with 5% CO 2 .

Techniques: Incubation, CCK-8 Assay, Staining, Fluorescence, Western Blot, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Gut Microbiota-Derived Propionic Acid Mediates ApoA-I-Induced Amelioration of MASLD via Activation of GPR43–Ca 2+ –CAMKII–ATGL Hepatic Lipolysis

doi: 10.3390/ijms27010468

Figure Lengend Snippet: PPA promotes activation of CAMKII and ATGL in a Ca 2 ⁺-dependent manner in OA-overloaded HepG2 cells. ( A ) After a 24 h incubation with 500 μM OA or BSA, HepG2 cells were exposed to 0.5 or 1 mM PPA for 48 h. Cytosolic Ca 2+ levels were measured. ( B , C ) Western blot analysis for detecting phosphorylated CAMKII (p-CAMKII) and total CAMKII (t-CAMKII). ( D – F ) HepG2 cells pretreated with OA or BSA for 24 h were co-incubated with PPA (0.5 or 1 mM) in the presence or absence of 10 μM BAPTA. Protein extracts were then assessed for p-CAMKII, t-CAMKII, p-ATGL, and t-ATGL via Western blotting. Groups are denoted by color: control (gray), OA alone (green), OA + PPA (orange), and OA + PPA + BAPTA (lime green). PPA concentrations are as indicated. All groups are labeled on the x-axis. Data are expressed as mean ± SEM; n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns represents no significant difference.

Article Snippet: The HepG2 human hepatoma cell line (obtained from ATCC) was maintained under standard conditions at 37 °C with 5% CO 2 .

Techniques: Activation Assay, Incubation, Western Blot, Control, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Gut Microbiota-Derived Propionic Acid Mediates ApoA-I-Induced Amelioration of MASLD via Activation of GPR43–Ca 2+ –CAMKII–ATGL Hepatic Lipolysis

doi: 10.3390/ijms27010468

Figure Lengend Snippet: GPR43 is involved in PPA-induced activation of the Ca 2 ⁺–CAMKII–ATGL pathway in HepG2 cells. ( A ) After a 24 h incubation with 500 μM OA or BSA, HepG2 cells were exposed to 0.5 or 1 mM PPA with or without 1 μM GLPG0974, after which cytosolic Ca 2 ⁺ levels were measured. ( B – D ) Protein extracts were assessed for p-CAMKII, t-CAMKII, p-ATGL and t-ATGL via Western blotting. Groups are denoted by color: control (gray), OA alone (green), OA + PPA (orange), and OA + PPA + GLPG0974 (lime green). PPA concentrations are as indicated. All groups are labeled on the x-axis. Data represent mean ± SEM; n = 3. * p < 0.05, ** p < 0.01; ns represents no significant difference.

Article Snippet: The HepG2 human hepatoma cell line (obtained from ATCC) was maintained under standard conditions at 37 °C with 5% CO 2 .

Techniques: Activation Assay, Incubation, Western Blot, Control, Labeling

Journal: bioRxiv

Article Title: HypoxamicroRNA-210 protects against hepatic steatosis by inhibiting CIDEC expression

doi: 10.64898/2025.12.19.695309

Figure Lengend Snippet: HepG2 cells were transfected with 1nM Control mimic (Ctrl) or miR-210 mimic, then treated with fatty acids and cultured under hypoxic conditions. miR-210 expression ( A ), intracellular triglyceride levels ( B ), and gene expression ( C ) were analyzed. ( D ) CIDEC protein expression was assessed by Western blotting. Quantification is shown in the histogram. (E-F) RNA pull-down assays. (E) Schematic illustration of the RNA pull-down experiment. Sequences of biotin-conjugated miR-210 mimic and the CIDEC mRNA 3’UTR region containing miR-210 binding site are shown. HepG2 cells were transfected with biotin-conjugated miR-210 mimic or control mimic, treated with fatty acids under hypoxic conditions. Streptavidin-coated magnetic beads captured biotin-labeled miR-210 and associated mRNAs via its binding sites in 3’ UTR. (F) qPCR analysis of miR-210, CIDEC and ACTB (negative control) mRNAs in the pull-down fraction. (G–H) Dual-luciferase reporter assay in HepG2 cells co-transfected with WT (G) or mutant (Mut, H ) CIDEC 3′UTR luciferase reporter and control or miR-210 mimic, followed by exposure to fatty acids and hypoxia. (I) Western blot validation of FLAG-CIDEC-GFP and FLAG-GFP expression using anti-CIDEC and anti-FLAG antibodies. α-tubulin was used as loading control. (J) Representative confocal images demonstrating FLAG-GFP or FLAG-CIDEC-GFP expression (green), lipid droplets stained with Oil Red O (red), and nuclei stained with DAPI (blue). Scale bar: 10 μm. (K) Quantification of intracellular triglyceride levels under the indicated conditions. Data are presented as mean ± SEM; n=3-6. Statistical significance was determined by unpaired Student’s t-test test or Mann-Whitney U test. * p < 0.05; ** p < 0.01. ns: no significant difference.

Article Snippet: Human hepatoma cell line HepG2 cells (ATCC, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, 4.5 g/L glucose) supplemented with 100 U/ml penicillin, 100 μg/mL streptomycin, and 10% FBS (ThermoFisher Scientific).

Techniques: Transfection, Control, Cell Culture, Expressing, Gene Expression, Western Blot, Binding Assay, Magnetic Beads, Labeling, Negative Control, Luciferase, Reporter Assay, Mutagenesis, Biomarker Discovery, Staining, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Long non-coding RNA enhances SARS-CoV-2-mediated apoptosis through epigenetic repression of angiotensin-converting enzyme 2

doi: 10.1016/j.jbc.2025.110812

Figure Lengend Snippet: SARS-CoV-2 and other coronaviruses consistently upregulate EAS1. A , RNA-seq analysis of EAS1 expression in Caco-2 cells infected with SARS-CoV-2 and Calu-3 cells infected with SARS-CoV at the indicated time points (n = 2 biologically independent samples). B , EAS1 expression in Calu-3, A549, and ACE2-overexpressing A549 (ACE2-A549) cells 24 h post-infection (hpi) with SARS-CoV-2 (n = 3). C , EAS1 levels in ACE2-A549 cells infected with SARS-CoV-2 (MOI = 0.1) for 24 h (n = 3). D , EAS1 expression in ACE2-A549 cells 24 hpi with SARS-CoV-2 (USA-WA1/2020) (n = 3). E , EAS1 levels in a blood vessel-liver organoid co-culture model 4 days post SARS-CoV-2 infection (n = 3). F , Time course of EAS1 expression in Calu-3 cells infected with SARS-CoV (Delta ORF6-mutant, MOI = 5) (n = 3). G , EAS1 expression in Calu-3 2B4 cells infected with MERS-CoV (HCoV-EMC) (n = 3). H , EAS1 levels in MRC5 cells infected with HCoV-229E (n = 3). I , EAS1 expression in A549 cells under hypoxia (1%O 2 ) for 42 h (n = 3). J , qPCR analysis of EAS1 in HepG2, A549, and immortalized HUVEC cells under hypoxia (1% O 2 ) for 24 and 48 h (n = 4 biologically independent experiments, each performed in triplicate). K , qPCR analysis of EAS1 in primary human hepatocytes, AT II cells, and HUVECs from four distinct donors under hypoxia (1% O 2 ) for 24 h (n = 3 independent experiments, each in triplicate). Data are mean ± SD. p values were determined by a two-tailed paired Student's t test, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: Human Hepatoma cell line HepG2 (ATCC HB-8065) and human lung cancer cell line A549 (ATCC CCL-185) were purchased from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco, 11995065) containing 10% fetal bovine serum (FBS, Biowest, S1810).

Techniques: RNA Sequencing, Expressing, Infection, Co-Culture Assay, Mutagenesis, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Long non-coding RNA enhances SARS-CoV-2-mediated apoptosis through epigenetic repression of angiotensin-converting enzyme 2

doi: 10.1016/j.jbc.2025.110812

Figure Lengend Snippet: HIF-1α mediates the induction of EAS1 by SARS-CoV-2. ( A ) HIF1A mRNA levels in A549 and ACE2-A549 cells 24 hpi with SARS-CoV-2 (n = 3); ( B ) HIF1A expression in ACE2-A549 cells infected with SARS-CoV-2 (USA-WA1/2020) for 24 h (n = 3); ( C ) HIF1A levels in Calu-3 cells infected with SARS-CoV-2 or SARS-CoV (n = 2); ( D ) HIF1A expression in ACE2-A549 cells infected with SARS-CoV-2 (MOI = 0.1) for 24 h (n = 3); ( E ) Pearson correlation analysis of HIF1A and EAS1 expression across human tissues from the GTEx database, results are shown as (Pearson Correlation Coefficient, p -value); ( F ) Western blot analysis of HIF-1α protein levels after knockdown (KD) or overexpression (OE) in HepG2, A549, and immortalized HUVEC cells (n = 3 independent experiments, each in twice); ( G ) qPCR analysis of EAS1 expression (n = 3, each in triplicate); ( H ) Enrichment levels of H3K27ac and H3K4me1 at EAS1 genomic loci and transcriptional activity analysis, data were extracted from the UCSC Genome Browser (GRCh38/hg38 assembly); ( I ) DNA-binding motif of HIF1A from the JASPAR database; ( J ) Schematic of the HIF1α-binding site within the first intron of EAS1, indicating locations for ChIP-qPCR primers and CRISPRi and CRISPRa sgRNA; ( K ) ChIP-qPCR analysis of HIF1α binding at the EAS1 enhancer following HIF1A KD or OE (n = 3, each in triplicate); ( L ) qPCR analysis of EAS1 expression after targeted epigenetic repression or activation (dCas9-VP64 or dCas9-KRAB) of the enhancer region (n = 3, each in triplicate). Data are mean ± SD. p values were determined by a two-tailed paired Student's t test, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: Human Hepatoma cell line HepG2 (ATCC HB-8065) and human lung cancer cell line A549 (ATCC CCL-185) were purchased from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco, 11995065) containing 10% fetal bovine serum (FBS, Biowest, S1810).

Techniques: Expressing, Infection, Western Blot, Knockdown, Over Expression, Activity Assay, Binding Assay, ChIP-qPCR, Activation Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Long non-coding RNA enhances SARS-CoV-2-mediated apoptosis through epigenetic repression of angiotensin-converting enzyme 2

doi: 10.1016/j.jbc.2025.110812

Figure Lengend Snippet: Inhibition of EAS1 attenuates SARS-CoV-2 infection-related apoptosis. A-B , flow cytometry analysis of apoptosis (Annexin V/PI staining) in HepG2, A549, and immortalized HUVEC cells treated with TNFα (HepG2-20ng, A549/immortalized HUVEC-10ng) for 24 h after EAS1 KD (n = 3). C-F , Western blot analysis of cleaved PARP1 and cleaved caspase 3 in EAS1 KD cells treated with TNFα (HepG2-20ng, A549/immortalized HUVEC-10ng), LPS (20 μg), or hypoxia (1%O 2 ) for 24 h (n = 3). G-K , Western blot analysis of apoptosis markers in primary human hepatocytes, AT II cells, and primary HUVECs with EAS1 KD under TNFα treatment for 24 h (representative of n = 1 experiment). L-O , Western blot analysis of apoptosis markers in EAS1 KD cells upon concomitant ACE2 KD and TNFα treatment (n = 3). Data are mean ± SD. Statistical significance was determined by a two-tailed paired Student's t test, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: Human Hepatoma cell line HepG2 (ATCC HB-8065) and human lung cancer cell line A549 (ATCC CCL-185) were purchased from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco, 11995065) containing 10% fetal bovine serum (FBS, Biowest, S1810).

Techniques: Inhibition, Infection, Flow Cytometry, Staining, Western Blot, Two Tailed Test