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: 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: Biomaterials

Article Title: A novel Kupffer cell-targeting nanoparticle system to Mitigate alcohol-associated liver disease

doi: 10.1016/j.biomaterials.2025.123623

Figure Lengend Snippet: Release of DEX from NPs in 1X PBS for 21 days at 37°C and two different pH: 6.0 and 7.4; (a) PC-0.2+DEX; (b) PC-0.5+DEX; (c) PC-1+DEX: only PC-1+DEX showed significantly high pH-dependent release of DEX; n = 3 (d) cytocompatibility studies in (i) HepG2 cells and (ii) DTHP1 (macrophages) confirmed cytocompatibility of PC-1 and PC-1+INT NPs in both cell lines 24 h after treatment; n = 4.

Article Snippet: Human hepatocellular carcinoma cell line HepG2 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques:

Journal: Biomaterials

Article Title: A novel Kupffer cell-targeting nanoparticle system to Mitigate alcohol-associated liver disease

doi: 10.1016/j.biomaterials.2025.123623

Figure Lengend Snippet: Uptake of NPs by DTHP1 and HepG2 cells treated with 1 μg/ml LPS and 0.16 % (v/v) EtOH; (a) quantitative analysis of NP uptake at different concentrations, confirmed higher, dose-dependent uptake by DTHP1 cells.; n = 4 (b) fluorescence imaging of NP uptake also confirmed higher uptake in DTHP1 than in HepG2 cells: green-coumarin-6 loaded NPs, blue-nuclei (scale bar = 150 μm) (c) quantification of NP uptake after cells were pre-treated with free INT (0.1 μM and 1 μM); uptake of the PC-1+INT NPs reduced due to the free INT pretreatment, confirming Gpbar1 targeting capabilities of the NPs.

Article Snippet: Human hepatocellular carcinoma cell line HepG2 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Fluorescence, Imaging