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