Journal: Cell & Bioscience

Article Title: TRIM16 mediates YAP1 K63-linked ubiquitination to alleviate sepsis-induced acute liver injury through YAP/Nrf2 axis in mice

doi: 10.1186/s13578-025-01517-4

Figure Lengend Snippet: The protein expression of TRIM16 was downregulated in LPS-injured hepatocytes. A Cell viability was assessed in mouse primary hepatocytes(a) and liver cell lines BRL-3 A(b) using CCK-8 assay following LPS exposure. n = 5 in each group. B Representative western blots showing the effects of LPS treatment on the expression of TRIM16 in WRL68(a), L02(b), BRL-3 A(c) and mouse primary hepatocytes(d), and the quantitative analysis ( n = 3 independent experiments). C Representative immunofluorescence images depicting TRIM16 subcellular distribution in LPS-stimulated hepatocytes. Mouse primary hepatocytes(a) and BRL-3 A(b) (the scale, 50 μm). Data were expressed as mean ± SD, * P < 0.05, ** P < 0.01 vs. CON

Article Snippet: The cells were starved in serum-free medium for 12 h, and then were treated with different interventions, including the following groups: (i) CON, the cells were maintained in serum-free medium with the vehicle; (ii) LPS, the cells were treated with 0.1, 1, 5, 10, 20 μg/mL LPS (Solarbio, Beijing, China) for 24 h and 1 μg/mL LPS for 3, 6, 12, 24, 48 h; (iii) siTRIM16, The cells with TRIM16 knockdown were transfected with siRNA-TRIM16 (GenePharma, Shanghai, China) (80 pmol/mL) and TransIT-X2 (Mirus, Madison, USA) for 24 h, followed by culturing in fresh DMEM medium; (iv) siTRIM16 + LPS, the cells were treated with siRNA-TRIM16 for 24 h followed by treatment with 1 μg/mL LPS for 24 h; (v) OE-TRIM16, the cells were exposed with the overexpressing TRIM16 cDNA plasmids (GenePharma, Shanghai, China) (1.2 μg/mL) for 48 h and then the medium was changed; (vi) OE-TRIM16 + LPS, the cells pretreated with TRIM16 cDNA plasmids for 48 h followed by treatment with 1 μg/mL LPS for 24 h; (vii) siTRIM16 + LPS + Verteporfin, Following 48-hour transfection with TRIM16-targeting siRNA, cells were pretreated with verteporfin (VP, 1 μM; MCE, USA), a YAP inhibitor, for 1 h prior to LPS stimulation; (viii) OE-TRIM16 + LPS + VP, when transfected with TRIM16 cDNA plasmids for 24 h, the cells were exposed to 1 μM VP for 1 h prior to treatment with LPS; viv) siYAP1 + OE-TRIM16, Following 24-hour transfection with YAP1-targeting siRNA, cells pretreated with TRIM16 cDNA plasmids for 48 h; vv) siYAP1 + OE-TRIM16 + LPS, Following 24-hour transfection with YAP1-targeting siRNA, cells pretreated with TRIM16 cDNA plasmids for 48 h followed by treatment with 1 μg/mL LPS for 24 h. Primary hepatocytes were isolated from the livers of the WT mice at age of 8-weeks as previously described [ ].

Techniques: Expressing, CCK-8 Assay, Western Blot, Immunofluorescence

Journal: Cell & Bioscience

Article Title: TRIM16 mediates YAP1 K63-linked ubiquitination to alleviate sepsis-induced acute liver injury through YAP/Nrf2 axis in mice

doi: 10.1186/s13578-025-01517-4

Figure Lengend Snippet: TRIM16 overexpression restores LPS-induced hepatocyte injury in vitro. A , B Immunofluorescence images demonstrating TRIM16 localization in hepatocytes (BRL-3 A) with genetic modulation (siRNA knockdown or plasmid overexpression) following 24 h LPS challenge (1 µg/mL) (the scale, upper 100 μm/lower 50 μm). C Cell viability in TRIM16-modulated hepatocytes (siRNA knockdown or plasmid overexpression) assessed using CCK-8 assay following 24 h LPS challenge (1 µg/mL). n = 5 in each group. ALT and AST levels in culture supernatants were quantified across experimental groups using enzymatic assays, with treatment conditions identical to those described in (A, B). n = 6 from 3 independent experiments. D The contents of SOD and MDA in hepatocytes with TRIM16 knockdown or overexpression under LPS stress. n = 5 in each group. E Quantitative analysis of TNF-α and IL-6 mRNA expression in hepatocytes performed using RT-qPCR ( n = 3 independent experiments). Data were expressed as mean ± SD, * P < 0.05, ** P < 0.01 vs. NC, # P < 0.05, ## P < 0.01 vs. NC + LPS.

Article Snippet: The cells were starved in serum-free medium for 12 h, and then were treated with different interventions, including the following groups: (i) CON, the cells were maintained in serum-free medium with the vehicle; (ii) LPS, the cells were treated with 0.1, 1, 5, 10, 20 μg/mL LPS (Solarbio, Beijing, China) for 24 h and 1 μg/mL LPS for 3, 6, 12, 24, 48 h; (iii) siTRIM16, The cells with TRIM16 knockdown were transfected with siRNA-TRIM16 (GenePharma, Shanghai, China) (80 pmol/mL) and TransIT-X2 (Mirus, Madison, USA) for 24 h, followed by culturing in fresh DMEM medium; (iv) siTRIM16 + LPS, the cells were treated with siRNA-TRIM16 for 24 h followed by treatment with 1 μg/mL LPS for 24 h; (v) OE-TRIM16, the cells were exposed with the overexpressing TRIM16 cDNA plasmids (GenePharma, Shanghai, China) (1.2 μg/mL) for 48 h and then the medium was changed; (vi) OE-TRIM16 + LPS, the cells pretreated with TRIM16 cDNA plasmids for 48 h followed by treatment with 1 μg/mL LPS for 24 h; (vii) siTRIM16 + LPS + Verteporfin, Following 48-hour transfection with TRIM16-targeting siRNA, cells were pretreated with verteporfin (VP, 1 μM; MCE, USA), a YAP inhibitor, for 1 h prior to LPS stimulation; (viii) OE-TRIM16 + LPS + VP, when transfected with TRIM16 cDNA plasmids for 24 h, the cells were exposed to 1 μM VP for 1 h prior to treatment with LPS; viv) siYAP1 + OE-TRIM16, Following 24-hour transfection with YAP1-targeting siRNA, cells pretreated with TRIM16 cDNA plasmids for 48 h; vv) siYAP1 + OE-TRIM16 + LPS, Following 24-hour transfection with YAP1-targeting siRNA, cells pretreated with TRIM16 cDNA plasmids for 48 h followed by treatment with 1 μg/mL LPS for 24 h. Primary hepatocytes were isolated from the livers of the WT mice at age of 8-weeks as previously described [ ].

Techniques: Over Expression, In Vitro, Immunofluorescence, Knockdown, Plasmid Preparation, CCK-8 Assay, Expressing, Quantitative RT-PCR

Journal: Cell & Bioscience

Article Title: TRIM16 mediates YAP1 K63-linked ubiquitination to alleviate sepsis-induced acute liver injury through YAP/Nrf2 axis in mice

doi: 10.1186/s13578-025-01517-4

Figure Lengend Snippet: TRIM16 promotes YAP nuclear translocation and YAP/Nrf2 protein expression. A , B Representative western blot images showing the expression of YAP1, p-YAP1(Ser127), Nrf2, or TEAD1 in hepatocytes (BRL3A) transfected with TRIM16 siRNA or overexpressed plasmids, challenged by LPS (1 µg/mL) for 24 h, and the quantitative analysis ( n = 3 independent experiments). C , D Representative fluorescence images of the distribution of YAP1 in hepatocytes in different groups (the scale, 50 μm). Data were expressed as mean ± SD, * P < 0.05, ** P < 0.01 vs. NC, # P < 0.05, ## P < 0.01 vs. NC + LPS

Article Snippet: The cells were starved in serum-free medium for 12 h, and then were treated with different interventions, including the following groups: (i) CON, the cells were maintained in serum-free medium with the vehicle; (ii) LPS, the cells were treated with 0.1, 1, 5, 10, 20 μg/mL LPS (Solarbio, Beijing, China) for 24 h and 1 μg/mL LPS for 3, 6, 12, 24, 48 h; (iii) siTRIM16, The cells with TRIM16 knockdown were transfected with siRNA-TRIM16 (GenePharma, Shanghai, China) (80 pmol/mL) and TransIT-X2 (Mirus, Madison, USA) for 24 h, followed by culturing in fresh DMEM medium; (iv) siTRIM16 + LPS, the cells were treated with siRNA-TRIM16 for 24 h followed by treatment with 1 μg/mL LPS for 24 h; (v) OE-TRIM16, the cells were exposed with the overexpressing TRIM16 cDNA plasmids (GenePharma, Shanghai, China) (1.2 μg/mL) for 48 h and then the medium was changed; (vi) OE-TRIM16 + LPS, the cells pretreated with TRIM16 cDNA plasmids for 48 h followed by treatment with 1 μg/mL LPS for 24 h; (vii) siTRIM16 + LPS + Verteporfin, Following 48-hour transfection with TRIM16-targeting siRNA, cells were pretreated with verteporfin (VP, 1 μM; MCE, USA), a YAP inhibitor, for 1 h prior to LPS stimulation; (viii) OE-TRIM16 + LPS + VP, when transfected with TRIM16 cDNA plasmids for 24 h, the cells were exposed to 1 μM VP for 1 h prior to treatment with LPS; viv) siYAP1 + OE-TRIM16, Following 24-hour transfection with YAP1-targeting siRNA, cells pretreated with TRIM16 cDNA plasmids for 48 h; vv) siYAP1 + OE-TRIM16 + LPS, Following 24-hour transfection with YAP1-targeting siRNA, cells pretreated with TRIM16 cDNA plasmids for 48 h followed by treatment with 1 μg/mL LPS for 24 h. Primary hepatocytes were isolated from the livers of the WT mice at age of 8-weeks as previously described [ ].

Techniques: Translocation Assay, Expressing, Western Blot, Transfection, Fluorescence

Journal: Cell & Bioscience

Article Title: TRIM16 mediates YAP1 K63-linked ubiquitination to alleviate sepsis-induced acute liver injury through YAP/Nrf2 axis in mice

doi: 10.1186/s13578-025-01517-4

Figure Lengend Snippet: TRIM16 mitigated LPS-induced hepatocyte cell injury via YAP1/Nrf2 pathways. A Hepatocyte (BRL-3 A) viability was assessed using CCK-8 assay following combinatorial treatment with verteporfin (1 µM) and LPS (1 µg/mL) for 24 h. n = 5 in each group. B Representative western blot images and the quantitative summary showing that YAP1 inhibitor, verteporfin inhibited the protein expression of YAP1 and Nrf2 ( n = 3 independent experiments). C Representative western blot images and the quantitative data showing that verteporfin abrogated the elevated effect of OE-TRIM16 and worsened the reduced effect of siTRIM16 on Nrf2 levels ( n = 3 independent experiments). D LDH levels in the supernatant and SOD and MDA levels of cultured cells upon verteporfin pretreatment. n = 6 from 3 independent experiments. E Measurements of Nrf2 mRNA in BRL-3 A cells using RT-qPCR analysis. n = 5. F Representative fluorescence images of the distribution of Nrf2 in hepatocytes in different groups (the scale, 40 μm). G Representative western blot images and the quantitative data showing that silencing YAP1 abrogated the elevated effect of OE-TRIM16 on Nrf2 levels ( n = 3 independent experiments). Data were expressed as mean ± SD, * P < 0.05, ** P < 0.01 vs. NC, # P < 0.05, ## P < 0.01 vs. NC + LPS, & P < 0.05, && P < 0.01 vs. siTRIM16 + LPS

Article Snippet: The cells were starved in serum-free medium for 12 h, and then were treated with different interventions, including the following groups: (i) CON, the cells were maintained in serum-free medium with the vehicle; (ii) LPS, the cells were treated with 0.1, 1, 5, 10, 20 μg/mL LPS (Solarbio, Beijing, China) for 24 h and 1 μg/mL LPS for 3, 6, 12, 24, 48 h; (iii) siTRIM16, The cells with TRIM16 knockdown were transfected with siRNA-TRIM16 (GenePharma, Shanghai, China) (80 pmol/mL) and TransIT-X2 (Mirus, Madison, USA) for 24 h, followed by culturing in fresh DMEM medium; (iv) siTRIM16 + LPS, the cells were treated with siRNA-TRIM16 for 24 h followed by treatment with 1 μg/mL LPS for 24 h; (v) OE-TRIM16, the cells were exposed with the overexpressing TRIM16 cDNA plasmids (GenePharma, Shanghai, China) (1.2 μg/mL) for 48 h and then the medium was changed; (vi) OE-TRIM16 + LPS, the cells pretreated with TRIM16 cDNA plasmids for 48 h followed by treatment with 1 μg/mL LPS for 24 h; (vii) siTRIM16 + LPS + Verteporfin, Following 48-hour transfection with TRIM16-targeting siRNA, cells were pretreated with verteporfin (VP, 1 μM; MCE, USA), a YAP inhibitor, for 1 h prior to LPS stimulation; (viii) OE-TRIM16 + LPS + VP, when transfected with TRIM16 cDNA plasmids for 24 h, the cells were exposed to 1 μM VP for 1 h prior to treatment with LPS; viv) siYAP1 + OE-TRIM16, Following 24-hour transfection with YAP1-targeting siRNA, cells pretreated with TRIM16 cDNA plasmids for 48 h; vv) siYAP1 + OE-TRIM16 + LPS, Following 24-hour transfection with YAP1-targeting siRNA, cells pretreated with TRIM16 cDNA plasmids for 48 h followed by treatment with 1 μg/mL LPS for 24 h. Primary hepatocytes were isolated from the livers of the WT mice at age of 8-weeks as previously described [ ].

Techniques: CCK-8 Assay, Western Blot, Expressing, Cell Culture, Quantitative RT-PCR, Fluorescence

Journal: Cell & Bioscience

Article Title: TRIM16 mediates YAP1 K63-linked ubiquitination to alleviate sepsis-induced acute liver injury through YAP/Nrf2 axis in mice

doi: 10.1186/s13578-025-01517-4

Figure Lengend Snippet: TRIM16 overexpression promotes YAP1 K63-linked ubiquitination and stabilizes its function. A Measurements of TRIM16 and YAP1 mRNA in BRL-3 A cells using RT-qPCR analysis. n = 5. B BRL-3 A cells were transfected with OE-TRIM16 plasmid and subsequently exposed to LPS. Protein-protein interaction was determined by western immunoprecipitation. Representative western blot images showing that OE-TRIM16 partially restored the decrease in the interaction between YAP1 and TRIM16 after LPS treatment in hepatocytes ( n = 3 independent experiments). C Cultured BRL-3 A cells were immunostained with anti-YAP1 antibody (green), anti-TRIM16 antibody (red), and DAPI nuclear counterstain (scale bar, 10 μm). Representative fluorescence micrographs showing the subcellular localization patterns of both proteins, with TRIM16-YAP1 colocalization quantitatively analyzed using Spearman’s correlation coefficient based on merged image pixel intensities. ( n = 30 cells from 3 independent experiments). Data were expressed as mean ± SD, * P < 0.05, ** P < 0.01 vs. NC, # P < 0.05, ## P < 0.01 vs. NC + LPS

Article Snippet: The cells were starved in serum-free medium for 12 h, and then were treated with different interventions, including the following groups: (i) CON, the cells were maintained in serum-free medium with the vehicle; (ii) LPS, the cells were treated with 0.1, 1, 5, 10, 20 μg/mL LPS (Solarbio, Beijing, China) for 24 h and 1 μg/mL LPS for 3, 6, 12, 24, 48 h; (iii) siTRIM16, The cells with TRIM16 knockdown were transfected with siRNA-TRIM16 (GenePharma, Shanghai, China) (80 pmol/mL) and TransIT-X2 (Mirus, Madison, USA) for 24 h, followed by culturing in fresh DMEM medium; (iv) siTRIM16 + LPS, the cells were treated with siRNA-TRIM16 for 24 h followed by treatment with 1 μg/mL LPS for 24 h; (v) OE-TRIM16, the cells were exposed with the overexpressing TRIM16 cDNA plasmids (GenePharma, Shanghai, China) (1.2 μg/mL) for 48 h and then the medium was changed; (vi) OE-TRIM16 + LPS, the cells pretreated with TRIM16 cDNA plasmids for 48 h followed by treatment with 1 μg/mL LPS for 24 h; (vii) siTRIM16 + LPS + Verteporfin, Following 48-hour transfection with TRIM16-targeting siRNA, cells were pretreated with verteporfin (VP, 1 μM; MCE, USA), a YAP inhibitor, for 1 h prior to LPS stimulation; (viii) OE-TRIM16 + LPS + VP, when transfected with TRIM16 cDNA plasmids for 24 h, the cells were exposed to 1 μM VP for 1 h prior to treatment with LPS; viv) siYAP1 + OE-TRIM16, Following 24-hour transfection with YAP1-targeting siRNA, cells pretreated with TRIM16 cDNA plasmids for 48 h; vv) siYAP1 + OE-TRIM16 + LPS, Following 24-hour transfection with YAP1-targeting siRNA, cells pretreated with TRIM16 cDNA plasmids for 48 h followed by treatment with 1 μg/mL LPS for 24 h. Primary hepatocytes were isolated from the livers of the WT mice at age of 8-weeks as previously described [ ].

Techniques: Over Expression, Ubiquitin Proteomics, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Cell Culture, Fluorescence

Journal: Cell & Bioscience

Article Title: TRIM16 mediates YAP1 K63-linked ubiquitination to alleviate sepsis-induced acute liver injury through YAP/Nrf2 axis in mice

doi: 10.1186/s13578-025-01517-4

Figure Lengend Snippet: TRIM16 overexpression improves CLP-induced liver injury by attenuating inflammation and oxidative stress in CLP-operated mice. A , B Representative images of HE and MASSON staining showing histopathological changes, including irregular cell arrangement, inflammatory infiltration (yellow arrows) and interstitial fibrosis (the scale, 50 μm/100 µm). C The changes of serum ALT and AST, and SOD, CAT and GSH in the livers of mice were measured. n = 6. D Quantitative data of IL-1β, TNF-α, and IL-6 mRNA in the liver tissues of CLP-operated mice ( n = 3 independent experiments). (E) Representative images of TUNEL staining showing apoptosis in the livers of mice in different groups (scale bar, 50 μm). Data were expressed as mean ± SD, * P < 0.05, ** P < 0.01 vs. NC, # P < 0.05, ## P < 0.01 vs. NC + CLP

Article Snippet: The cells were starved in serum-free medium for 12 h, and then were treated with different interventions, including the following groups: (i) CON, the cells were maintained in serum-free medium with the vehicle; (ii) LPS, the cells were treated with 0.1, 1, 5, 10, 20 μg/mL LPS (Solarbio, Beijing, China) for 24 h and 1 μg/mL LPS for 3, 6, 12, 24, 48 h; (iii) siTRIM16, The cells with TRIM16 knockdown were transfected with siRNA-TRIM16 (GenePharma, Shanghai, China) (80 pmol/mL) and TransIT-X2 (Mirus, Madison, USA) for 24 h, followed by culturing in fresh DMEM medium; (iv) siTRIM16 + LPS, the cells were treated with siRNA-TRIM16 for 24 h followed by treatment with 1 μg/mL LPS for 24 h; (v) OE-TRIM16, the cells were exposed with the overexpressing TRIM16 cDNA plasmids (GenePharma, Shanghai, China) (1.2 μg/mL) for 48 h and then the medium was changed; (vi) OE-TRIM16 + LPS, the cells pretreated with TRIM16 cDNA plasmids for 48 h followed by treatment with 1 μg/mL LPS for 24 h; (vii) siTRIM16 + LPS + Verteporfin, Following 48-hour transfection with TRIM16-targeting siRNA, cells were pretreated with verteporfin (VP, 1 μM; MCE, USA), a YAP inhibitor, for 1 h prior to LPS stimulation; (viii) OE-TRIM16 + LPS + VP, when transfected with TRIM16 cDNA plasmids for 24 h, the cells were exposed to 1 μM VP for 1 h prior to treatment with LPS; viv) siYAP1 + OE-TRIM16, Following 24-hour transfection with YAP1-targeting siRNA, cells pretreated with TRIM16 cDNA plasmids for 48 h; vv) siYAP1 + OE-TRIM16 + LPS, Following 24-hour transfection with YAP1-targeting siRNA, cells pretreated with TRIM16 cDNA plasmids for 48 h followed by treatment with 1 μg/mL LPS for 24 h. Primary hepatocytes were isolated from the livers of the WT mice at age of 8-weeks as previously described [ ].

Techniques: Over Expression, Staining, TUNEL Assay

Journal: Cell & Bioscience

Article Title: TRIM16 mediates YAP1 K63-linked ubiquitination to alleviate sepsis-induced acute liver injury through YAP/Nrf2 axis in mice

doi: 10.1186/s13578-025-01517-4

Figure Lengend Snippet: TRIM16 overexpression protected against CLP-induced liver injury through YAP/Nrf2 axis. Quantitative analysis of TRIM16 mRNA expression in the liver tissues of the septic mice. (B) Representative western blot images showing the expression of YAP1, Nrf2, and TRIM16 in the liver tissues of the septic mice, and the quantitative analysis ( n = 3 independent experiments). C Representative western blot images showing that TRIM16 overexpression partially restored the decrease in the interaction between YAP1 and TRIM16 after CLP insults in the livers of mice. Data were expressed as mean ± SD, * P < 0.05, ** P < 0.01 vs. NC, # P < 0.05, ## P < 0.01 vs. NC + CLP

Article Snippet: The cells were starved in serum-free medium for 12 h, and then were treated with different interventions, including the following groups: (i) CON, the cells were maintained in serum-free medium with the vehicle; (ii) LPS, the cells were treated with 0.1, 1, 5, 10, 20 μg/mL LPS (Solarbio, Beijing, China) for 24 h and 1 μg/mL LPS for 3, 6, 12, 24, 48 h; (iii) siTRIM16, The cells with TRIM16 knockdown were transfected with siRNA-TRIM16 (GenePharma, Shanghai, China) (80 pmol/mL) and TransIT-X2 (Mirus, Madison, USA) for 24 h, followed by culturing in fresh DMEM medium; (iv) siTRIM16 + LPS, the cells were treated with siRNA-TRIM16 for 24 h followed by treatment with 1 μg/mL LPS for 24 h; (v) OE-TRIM16, the cells were exposed with the overexpressing TRIM16 cDNA plasmids (GenePharma, Shanghai, China) (1.2 μg/mL) for 48 h and then the medium was changed; (vi) OE-TRIM16 + LPS, the cells pretreated with TRIM16 cDNA plasmids for 48 h followed by treatment with 1 μg/mL LPS for 24 h; (vii) siTRIM16 + LPS + Verteporfin, Following 48-hour transfection with TRIM16-targeting siRNA, cells were pretreated with verteporfin (VP, 1 μM; MCE, USA), a YAP inhibitor, for 1 h prior to LPS stimulation; (viii) OE-TRIM16 + LPS + VP, when transfected with TRIM16 cDNA plasmids for 24 h, the cells were exposed to 1 μM VP for 1 h prior to treatment with LPS; viv) siYAP1 + OE-TRIM16, Following 24-hour transfection with YAP1-targeting siRNA, cells pretreated with TRIM16 cDNA plasmids for 48 h; vv) siYAP1 + OE-TRIM16 + LPS, Following 24-hour transfection with YAP1-targeting siRNA, cells pretreated with TRIM16 cDNA plasmids for 48 h followed by treatment with 1 μg/mL LPS for 24 h. Primary hepatocytes were isolated from the livers of the WT mice at age of 8-weeks as previously described [ ].

Techniques: Over Expression, Expressing, Western Blot