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: FoxO transcription factors couple the urea cycle and gluconeogenesis by controlling Ass1

doi: 10.64898/2025.12.22.695746

Figure Lengend Snippet: ( A ) Top, the results of ChIP-seq with anti-FoxO1 antibody in livers of unfed mice obtained from ChIP-Atlas (ID: GSM3381273); bottom, simplified representation of the structure of the full fragment, fragment 1, fragment 2, fragment 3, and the subdivisions of fragment 1 i.e. small fragment 1A and small fragment 1B. ( B and C ) Enhancer analysis of FoxO1 and FoxO3a on Ass1 enhancer and its fragments. FoxO1 and FoxO3a expression plasmids were co-transfected with the indicated Ass1 enhancer firefly luciferase reporter plasmids in HepG2 cells. ( D and E ) Electrophoretic mobility shift assay (EMSA) using radiolabeled probe for ( D ) the two JASPAR predicted FoxO binding sites (Figure S5) in the smallest fragment 1A from the Ass1 enhancer and FoxO binding site in the promoter of Pck1 as a positive control; ( E ) the wild-type (WT) and mutant (Mut) predicted FoxO binding site1 incubated with GST and GST-FoxO recombinant proteins. BS, binding site; WT, wild-type; Mut, mutant. ( F ) Enhancer analysis of FoxO1 and FoxO3a on mutant Ass1 enhancer and its fragments. FoxO1 and FoxO3a expression plasmids were co-transfected with the indicated Ass1 enhancer firefly luciferase reporter plasmids in HepG2 cells. All values are presented as the mean with error bars representing the SEM. Datasets were assessed by Student’s t -test for unpaired samples. The differences were considered to be significant if P < 0.05 (* P < 0.05 and ** P <0.01).

Article Snippet: HEK293 human embryonic kidney cells (RRID:CVCL_0045) and HepG2 human hepatoma cells (RRID: CVCL_0027) were distributed from ATCC (American Type Culture Collection, Manassas, VA, USA), and cultured in DMEM containing 25 mM glucose, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate supplemented with 10% FBS.

Techniques: ChIP-sequencing, Expressing, Transfection, Luciferase, Electrophoretic Mobility Shift Assay, Binding Assay, Positive Control, Mutagenesis, Incubation, Recombinant, IF-P

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: International Journal of Molecular Sciences

Article Title: An ATF3 Inducer Ameliorates Metabolic Dysfunction-Associated Steatotic Liver Disease Through the AMPK and PKA Pathways

doi: 10.3390/ijms262411877

Figure Lengend Snippet: Effects of the ATF3 inducer, ST32db, on the viability of HepG2 cells. ( A ) The chemical structure of ST32db. ( B – D ) Cell viability was measured after the treatment with various concentrations of ST32db for 24 h, 48 h, and 72 h using a CCK8 assay. Data are mean ± SEM ( n = 4) and analyzed by one-way ANOVA. ## p < 0.01 compared to the control group.

Article Snippet: Human hepatoma HepG2 cells were obtained from ATCC (HB-8065, ATCC, Manassas, VA, USA).

Techniques: CCK-8 Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: An ATF3 Inducer Ameliorates Metabolic Dysfunction-Associated Steatotic Liver Disease Through the AMPK and PKA Pathways

doi: 10.3390/ijms262411877

Figure Lengend Snippet: ST32db decreased lipid accumulation in oleic acid (OA)-treated HepG2 cells. Cells were treated with OA for 24 h, followed by ST32db treatment for 24 h or 48 h. ( A ) Representative images (200× magnification; scale bar, 200 µm) showing OA-induced lipid accumulation visualized by Oil Red O staining. ( B , C ) The relative quantification of lipid content. Data are mean ± SEM ( n = 3) and analyzed by one-way ANOVA. ## p < 0.01 compared to control group; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to OA group; and $ p < 0.05 compared to 1 mM OA+20 uM ST32db.

Article Snippet: Human hepatoma HepG2 cells were obtained from ATCC (HB-8065, ATCC, Manassas, VA, USA).

Techniques: Staining, Quantitative Proteomics, Control

Journal: International Journal of Molecular Sciences

Article Title: An ATF3 Inducer Ameliorates Metabolic Dysfunction-Associated Steatotic Liver Disease Through the AMPK and PKA Pathways

doi: 10.3390/ijms262411877

Figure Lengend Snippet: ST32db regulated adipogenesis-, lipogenesis- and lipolysis-related gene and protein levels in OA-treated HepG2 cells. ( A ) Real-time PCR analysis of mRNA levels of ATF3 , adipogenic ( C/EBPα , C/EBPβ , PPARγ2) , lipogenic ( ACC , ChREBP , FAS , SCD1 , SREBP1 , DGAT1 , and DGAT2 ), and lipolytic ( ATGL ) genes after 48 h co-treatment with OA and ST32db normalized to GAPDH and relative to control. ( B ) The levels of ATF3, C/EBPβ, ChREBP, FAS, SREBP1, and ATGL proteins were measured by Western blot. ( C ) Densitometry quantification of Western blot. Data are presented as mean ± SEM ( n = 3~7) and analyzed by one-way ANOVA. # p < 0.05 and ## p < 0.01 compared to control; * p < 0.05 and ** p < 0.01 compared to OA group.

Article Snippet: Human hepatoma HepG2 cells were obtained from ATCC (HB-8065, ATCC, Manassas, VA, USA).

Techniques: Real-time Polymerase Chain Reaction, Control, Western Blot

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