Journal: Redox Biology

Article Title: UFMylation deficiency in hepatocytes activates the KEAP1-NRF2 pathway and contributes to hepatocarcinogenesis

doi: 10.1016/j.redox.2026.104046

Figure Lengend Snippet: Depletion of UFL1 causes aberrant oxidative stress and NRF2-ARE pathway activation. (A ) WikiPathways analysis of significantly differentially expressed genes in liver tissues from 3-month-old CKO vs. WT mice. This analysis revealed significant enrichment of several pathways associated with oxidative stress or metabolism. (B) Gene Set Enrichment Analysis (GSEA) of the Nrf2 pathway using RNA-seq data from liver tissues of 3-month-old CKO vs. WT mice. The normalized enrichment score (NES), nominal p-value, and false discovery rate (FDR) q-value were used to assess the significance of the association between gene sets and the signaling pathway. Criteria for statistical significance were set as |NES| > 1, FDR ≤0.25, and P < 0.05. (C) Expression levels of Nrf2 target genes in liver tissues were quantified by qPCR. Error bars represent the mean ± SD. ∗, P < 0.05; ∗∗, P < 0.01; ns, not significant. (D) Schematic of the NRF2-responsive reporter construct. Fluorescence intensity was visualized using fluorescence microscopy. Scale bars: 100 μm. (E) HepG2 cells transfected with the ARE-GFP reporter were verified by Western blot using the indicated antibodies. (F) Immunofluorescence staining for NRF2 (green) in cells following UFL1 knockdown or exogenous UFL1 rescue, respectively. Cell nuclei were counterstained with DAPI, and merged images show colocalization of NRF2 with DAPI. Scale bar: 50 μm. (G) Quantification of the NRF2 nuclear/cytoplasmic ratio. This ratio reflects the proportion of NRF2 protein localized in the nucleus relative to that in the cytoplasm, based on 30 randomly selected cells counted. Error bars represent the mean ± SD. ∗∗, P < 0.01; ∗∗∗, P < 0.001; ns, not significant.

Article Snippet: Human embryonic kidney cell line HEK293T and hepatocellular carcinoma cell line HepG2 cell lines used in this study were purchased from the American Type Culture Collection (ATCC) and grown in Dulbecco's modified Eagle medium (01-055-1A, Biological Industries) supplemented with 10 % fetal bovine serum (FSP500, ExCell Bio, Shanghai, China) and penicillin-streptomycin (15140-122, Gibco).

Techniques: Activation Assay, RNA Sequencing, Expressing, Construct, Fluorescence, Microscopy, Transfection, Western Blot, Immunofluorescence, Staining, Knockdown

Journal: Redox Biology

Article Title: UFMylation deficiency in hepatocytes activates the KEAP1-NRF2 pathway and contributes to hepatocarcinogenesis

doi: 10.1016/j.redox.2026.104046

Figure Lengend Snippet: Loss of UFL1 leads to activation of the NRF2-ARE pathway in hepatic cells. (A) Western blot analysis of the expression of Keap1, Nrf2, p62, Nqo1, SLC7A11, HO-1, and Gpx4 proteins in the livers of 3-month-old WT and CKO mice. (B) Western blot analysis of the expression of KEAP1, NRF2, p62, UFL1, and DDRGK1 proteins in HepG2 cells with UFL1 knockdown or control cells. (C) Western blot analysis of the expression of KEAP1, NRF2, UFM1, and UFSP2 proteins in HepG2 cells with UFSP2 knockdown or control cells. (D) Western blot analysis of the expression and subcellular localization of NRF2 in HepG2 cells with UFL1 knockdown or control cells. Calnexin, Histone H3, and GAPDH were used as loading control, respectively. (E) Immunohistochemical staining of Nrf2 in liver sections from 3-month-old WT and CKO mice. The larger dashed-boxed images below their corresponding overview images in the Nrf2 staining panel show higher-magnification views of Nrf2 nuclear staining. Scale bars: 100 μm. (F) Quantification of the number of cells with Nrf2 nuclear localization from Nrf2 immunohistochemical staining in liver sections from 3-month-old WT and CKO mice. Quantification was carried out from three WT and three CKO mice, at least 400 cells counted per mouse. Error bars represent the mean ± SD. ∗∗, P < 0.01.

Article Snippet: Human embryonic kidney cell line HEK293T and hepatocellular carcinoma cell line HepG2 cell lines used in this study were purchased from the American Type Culture Collection (ATCC) and grown in Dulbecco's modified Eagle medium (01-055-1A, Biological Industries) supplemented with 10 % fetal bovine serum (FSP500, ExCell Bio, Shanghai, China) and penicillin-streptomycin (15140-122, Gibco).

Techniques: Activation Assay, Western Blot, Expressing, Knockdown, Control, Immunohistochemical staining, Staining

Journal: Redox Biology

Article Title: UFMylation deficiency in hepatocytes activates the KEAP1-NRF2 pathway and contributes to hepatocarcinogenesis

doi: 10.1016/j.redox.2026.104046

Figure Lengend Snippet: UFMylation stabilizes KEAP1 by antagonizing its ubiquitination. (A) UFMylation assay of endogenous KEAP1 in HepG2 cells. HepG2 whole-cell lysates were subjected to co-immunoprecipitation (Co-IP) with KEAP1 (Ab) antibody or control IgG, followed by Western blot. Small black arrowheads indicate bands corresponding to covalently linked KEAP1-UFM1. (B, C) Analysis of the interaction between KEAP1 and UFL1 in HEK293T cells. HEK293T cell lysates were subjected to immunoprecipitation with anti-HA ( B ) or anti-FLAG ( C ) affinity gel, followed by Western blot to detect their mutual interaction. NRF2 and DDRGK1 served as positive controls for binding to KEAP1 and UFL1, respectively. (D) KEAP1 UFMylation was verified by Western blot following immunoprecipitation in UFSP1 KO /UFSP2 KO HEK293T cells expressing exogenous UFMylation system components and FLAG-tagged KEAP1. (E) KEAP1 ubiquitination was analyzed by Western blot in UFSP1 KO /UFSP2 KO HEK293T cells transfected with His-tagged ubiquitin (His-Ub) alone or with His-Ub plus UFMylation components. (F) KEAP1 protein stability was assessed by Western blot in UFL1-knockdown (sgUFL1) versus control HepG2 (sgCtrl) cells. Cells were treated with 100 μg/mL CHX for the indicated time. (G) Quantification of KEAP1 protein levels (corresponding to F ) is shown in the graph. Error bars represent the mean ± SD. ∗, P < 0.05.

Article Snippet: Human embryonic kidney cell line HEK293T and hepatocellular carcinoma cell line HepG2 cell lines used in this study were purchased from the American Type Culture Collection (ATCC) and grown in Dulbecco's modified Eagle medium (01-055-1A, Biological Industries) supplemented with 10 % fetal bovine serum (FSP500, ExCell Bio, Shanghai, China) and penicillin-streptomycin (15140-122, Gibco).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Western Blot, Binding Assay, Expressing, Transfection, Knockdown

Journal: Biotechnology Reports

Article Title: Insulin Resistance, Anti-inflammatory, and Antioxidant In vitro Activity of Heterologously Expressed Arenin from Dryophytes arenicolor

doi: 10.1016/j.btre.2026.e00947

Figure Lengend Snippet: In vitro modulation of hepatic insulin resistance by arenin in hepatic insulin-resistant (HepG2) cells . Arenin concentrations (3.90–1000 µg/mL) were incubated for 5 h in hepatic insulin-resistant cells. Negative control (N) cells were cultured in the absence of insulin, whereas insulin-resistant (IR) control cells were cultured in the presence of insulin. Rosiglitazone (ROSI) at 10 μM (3.57 µg/mL) was used as the positive reference control. Different letters abc indicate statistical differences between concentrations analyzed using Tukey test ( p < 0.05).

Article Snippet: Human hepatocyte carcinoma (HepG2) cells, human primary dermal fibroblasts (HDFa) cells, and murine macrophage (RAW 264.7) cells were obtained from the American Type Culture Collection (ATCC, VA, USA).

Techniques: In Vitro, Incubation, Negative Control, Cell Culture, Control

Journal: Medical Oncology (Northwood, London, England)

Article Title: Ashwagandha ( Withania somnifera ) targets liver cancer stem cells via inhibiting Hedgehog signaling pathway in hepatocellular carcinoma

doi: 10.1007/s12032-025-03215-0

Figure Lengend Snippet: Bar chart representing the viability (%) of HepG2 cells treated with different concentrations of sorafenib and/or ashwagandha aqueous extract for 48 h. *: Statistically significant difference from control group at p ≤ 0.05

Article Snippet: The present study was an in vitro study employing the human hepatocellular carcinoma cell line HepG2 obtained from ATCC (HB-8065 TM).

Techniques: Control

Journal: Medical Oncology (Northwood, London, England)

Article Title: Ashwagandha ( Withania somnifera ) targets liver cancer stem cells via inhibiting Hedgehog signaling pathway in hepatocellular carcinoma

doi: 10.1007/s12032-025-03215-0

Figure Lengend Snippet: Combination index plot for SOR and ASH-AE in HepG2 cells

Article Snippet: The present study was an in vitro study employing the human hepatocellular carcinoma cell line HepG2 obtained from ATCC (HB-8065 TM).

Techniques:

Journal: Medical Oncology (Northwood, London, England)

Article Title: Ashwagandha ( Withania somnifera ) targets liver cancer stem cells via inhibiting Hedgehog signaling pathway in hepatocellular carcinoma

doi: 10.1007/s12032-025-03215-0

Figure Lengend Snippet: Flow cytometry analysis of CD90 expression in HepG2 cells treated with SOR and/or ASH-AE for 48 h. *: Statistically significant difference from control group at p ≤ 0.05, #: Statistically significant difference from SOR group at p ≤ 0.05

Article Snippet: The present study was an in vitro study employing the human hepatocellular carcinoma cell line HepG2 obtained from ATCC (HB-8065 TM).

Techniques: Flow Cytometry, Expressing, Control

Journal: Medical Oncology (Northwood, London, England)

Article Title: Ashwagandha ( Withania somnifera ) targets liver cancer stem cells via inhibiting Hedgehog signaling pathway in hepatocellular carcinoma

doi: 10.1007/s12032-025-03215-0

Figure Lengend Snippet: Bar chart representing the effect of SOR and/or ASH-AE on gene expression of ( a ) SHh, ( b ) PATCH1and ( c ) ABCC1 in HepG2 cells. *: Statistically significant difference from control group at p ≤ 0.05, #: Statistically significant difference from SOR group at p ≤ 0.05

Article Snippet: The present study was an in vitro study employing the human hepatocellular carcinoma cell line HepG2 obtained from ATCC (HB-8065 TM).

Techniques: Gene Expression, Control

Journal: Medical Oncology (Northwood, London, England)

Article Title: Ashwagandha ( Withania somnifera ) targets liver cancer stem cells via inhibiting Hedgehog signaling pathway in hepatocellular carcinoma

doi: 10.1007/s12032-025-03215-0

Figure Lengend Snippet: Representative confocal immunofluorescence images of Hepg2 cells with immunoreactivity of Gli1 showing different positivity as following: ( A ) Strong positive (↑) immune reaction in untreated HepG2 cells, ( B ) Strong positive immune reaction in HepG2 cells treated with SOR. Note: translocated Gli1 to the nucleus (↑), ( C ) Weak positive (↑) immune reaction in HepG2 cells treated with ASH-AE, ( D ) Moderate positive (↑) immune reaction in HepG2 cells treated with SOR + ASH-AE

Article Snippet: The present study was an in vitro study employing the human hepatocellular carcinoma cell line HepG2 obtained from ATCC (HB-8065 TM).

Techniques: Immunofluorescence

Journal: Medical Oncology (Northwood, London, England)

Article Title: Ashwagandha ( Withania somnifera ) targets liver cancer stem cells via inhibiting Hedgehog signaling pathway in hepatocellular carcinoma

doi: 10.1007/s12032-025-03215-0

Figure Lengend Snippet: Bar chart representing the effect of SOR and/or ASH-AE on the percentage of Gli1 nuclear translocation in HepG2 cells. *: Statistically significant difference from control group at p ≤ 0.05, #: Statistically significant difference from SOR group at p ≤ 0.05, ⁓: Statistically significant difference from ASH-AE group at p ≤ 0.05

Article Snippet: The present study was an in vitro study employing the human hepatocellular carcinoma cell line HepG2 obtained from ATCC (HB-8065 TM).

Techniques: Translocation Assay, Control

Journal: Chemmedchem

Article Title: Structure‐Based Discovery of Obeticholic Acid Derivatives as Novel Farnesoid X Receptor Partial Agonists with Improved Selectivity and Reduced Off‐Target Effects

doi: 10.1002/cmdc.202500960

Figure Lengend Snippet: Results of RT‐PCR in HepG2 cell line following overnight treatment with endogenous FXR agonist CDCA ( red ) (30 μM), OCA ( purple ) (10 µM), compound 2 ( green ) (10 μM and 30 μM), or compound 16 ( blue ) (10 μM and 30 μM). Not treated cells ( white ) were stimulated with DMSO 0.1. % Statistical significance was analyzed by One‐way Anova‐GraphPad Prism 5. ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001 vs 0.1% NT = not treated (DMSO).

Article Snippet: HepG2 human hepatocellular carcinoma cells (RRID:CVCL_0027, HB‐8065, ATCC) were cultured in E‐MEM medium supplemented with 1% penicillin/streptomycin, 1% L‐glutamine, and 10% fetal bovine serum (Life Technologies).

Techniques: Reverse Transcription Polymerase Chain Reaction