Journal: bioRxiv

Article Title: Development of a New Approach Method to Monitor and Modify Caffeine Metabolism Correlated to CYP1A2 Expression

doi: 10.1101/2025.11.27.691042

Figure Lengend Snippet:

Article Snippet: Human HCC cell lines Hep3B (HB-8064) and HepG2 (HB-8065) were purchased from ATCC (Manassas, VA, USA).

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Development of a New Approach Method to Monitor and Modify Caffeine Metabolism Correlated to CYP1A2 Expression

doi: 10.1101/2025.11.27.691042

Figure Lengend Snippet: Modulation of paraxanthine accumulation by known CYP1A2 effectors in HepG2 and Hep3B cells. Paraxanthine concentration was quantified by LC–MRM. Data are shown as mean ± SD from three independent experiments. A-B, Sulforaphane reduced paraxanthine accumulation in HepG2 and Hep3B cells. C-D , Galangin increased paraxanthine accumulation in both cell lines. E-F , 3-Methylcholanthrene (3-MC) also increased paraxanthine accumulation in both cell lines. The results of t-test are reported as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Human HCC cell lines Hep3B (HB-8064) and HepG2 (HB-8065) were purchased from ATCC (Manassas, VA, USA).

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Development of a New Approach Method to Monitor and Modify Caffeine Metabolism Correlated to CYP1A2 Expression

doi: 10.1101/2025.11.27.691042

Figure Lengend Snippet: qPCR measurement of CYP1A2 mRNA levels in HepG2 (top panels) and Hep3B (bottom panels) cells after 24-hour treatments with A-B , caffeine and caffeine along with C-D, D,L-sulforaphane, E-F , 3-methylcholanthrene, or G-H , galangin at the indicated concentrations. Control cells received the vehicle only. Data are normalized to a housekeeping gene and expressed as the mean ± SD (n = 3) from three independent experiments. The results of t-test are reported as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Human HCC cell lines Hep3B (HB-8064) and HepG2 (HB-8065) were purchased from ATCC (Manassas, VA, USA).

Techniques: Control

Journal: The Journal of Biological Chemistry

Article Title: O -GlcNAcylation of fatty acid synthase is required for its proper subcellular localization, expression level, and activity

doi: 10.1016/j.jbc.2025.110497

Figure Lengend Snippet: Mutation of the T980 O -GlcNAcylation site reduces FASN expression, membrane association, homodimerization, and stability . Hep3B cells were transfected with an empty vector or 3xFlag-FASN (wild type and mutants) expressing vectors. Cell lysates were analyzed by Western blot (n = 8) by using an anti-Flag antibody ( A ). Molecular mass markers are indicated on the left (kDa). Optical densities were measured and normalized with β-actin expression. B , cytosolic and membrane-associated 3xFlag-FASN contents were prepared by cell fractionation and analyzed by Western blot (n = 3). Fractionation efficiency was assessed using anti-GAPDH and anti-E-Cadherin antibodies for cytosolic and membrane fractions, respectively. Optical densities were measured and normalized with these markers. C , cell lysates were analyzed by Native-PAGE (n = 3) according to their 3xFlag-FASN monomers (∼250 kDa) and dimers (∼500 kDa) contents. Optical densities were measured and normalized with total protein. D , CHX chase assay was performed by treating cells with 40 μg/ml CHX for the indicated time periods (0–24 h). Data are presented with means ± SD. ∗p < 0.05; ∗∗ p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: HepG2 and Hep3B cell lines are ATCC certified.

Techniques: Mutagenesis, Expressing, Membrane, Transfection, Plasmid Preparation, Western Blot, Cell Fractionation, Fractionation, Clear Native PAGE

Journal: The Journal of Biological Chemistry

Article Title: O -GlcNAcylation of fatty acid synthase is required for its proper subcellular localization, expression level, and activity

doi: 10.1016/j.jbc.2025.110497

Figure Lengend Snippet: FASN T980 is crucial for FASN activity . Hep3B cells were transfected with an empty vector or 3xFlag-FASN (wild type and mutants) expressing vectors and incubated with the lipid droplet marker BODIPY 493/503 (n = 3) ( A ). Lipid droplets were counted using the ImageJ software ( B ). Confocal microscopy analysis indicates that FASN T980A mutation significantly impacts lipid droplets formation (bar size: 15 μm). Data are presented with means ± SD. ∗ p < 0.05.

Article Snippet: HepG2 and Hep3B cell lines are ATCC certified.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Incubation, Marker, Software, Confocal Microscopy, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: O -GlcNAcylation of fatty acid synthase is required for its proper subcellular localization, expression level, and activity

doi: 10.1016/j.jbc.2025.110497

Figure Lengend Snippet: FASN O -GlcNAcylation at T980 is pivotal for Hep3B cells survival, proliferation, and cell cycle progression. Low-density Hep3B cells were transfected with an empty vector or 3xFlag-FASN (wild type and mutants) expressing vectors. Six days later, colonies were fixed and stained with crystal violet (n = 3) ( A ). Using MTS assay ( B ), cell survival was evaluated according to densitometry measured at λ = 490 nm (n = 6). Cell proliferation was determined by cell counting (n = 4) ( C ). Cell cycle was analyzed by flow cytometry (n = 3) ( D ). Data are presented with means ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: HepG2 and Hep3B cell lines are ATCC certified.

Techniques: Transfection, Plasmid Preparation, Expressing, Staining, MTS Assay, Cell Counting, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: O -GlcNAcylation of fatty acid synthase is required for its proper subcellular localization, expression level, and activity

doi: 10.1016/j.jbc.2025.110497

Figure Lengend Snippet: FASN T980 is crucial for various properties of Hep3B cells. The O -GlcNAcylation at T980 is crucial for FASN expression, stability, membrane residence, homodimerization, and activity, promoting Hep3B cells survival, proliferation, and cell cycle progression. This highlights the O -GlcNAcylation of FASN at T980 as a potential key modification that supports hepatic carcinogenesis.

Article Snippet: HepG2 and Hep3B cell lines are ATCC certified.

Techniques: Expressing, Membrane, Activity Assay, Modification