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