Journal: Molecular Medicine Reports

Article Title: Pathological mechanism of ferroptosis in a rat model of α-naphthyl isothiocyanate-induced chronic cholestasis

doi: 10.3892/mmr.2026.13802

Figure Lengend Snippet: Abnormal expression of antioxidant and iron metabolism-related proteins in liver samples of rats with chronic cholestasis. Control group was fed a chow diet, whereas the ANIT and DFO-treated groups were intragastrically administered ANIT olive oil solution with or without DFO (n=8/group). (A) Changes in ferroptosis antioxidant-related protein expression. The blots shown are representative of three independent experiments. A dotted line indicates that the lanes were non-adjacent on the original gel. All target protein bands and their corresponding loading control bands shown side-by-side were derived from the same membrane. Relative expression levels of (B) Nrf2, (C) Keap1, (D) XCT, (E) HO-1 and (F) GPX4. (G) Changes in iron metabolism-related protein expression. The blots shown are representative of three independent experiments. A dotted line indicates that the lanes were non-adjacent on the original gel. All target protein bands and their corresponding loading control bands shown side-by-side were derived from the same membrane. Relative expression levels of (H) TFR1, (I) FPN1, (J) DMT1, (K) Steap3 and (L) FTH1. Data are presented as the mean ± SD. P-values were determined by one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001. ANIT, α-naphthyl isothiocyanate; DFO, deferoxamine; Nrf2, nuclear factor erythroid-2-related factor 2; Keap1, Kelch-like ECH-associated protein 1, XCT, cystine/glutamate transporter; HO-1, heme oxygenase 1; GPX4, glutathione peroxidase 4; TFR1, transferrin receptor 1; FPN1, ferroportin 1; DMT1, divalent metal transporter 1; Steap3, six-transmembrane epithelial antigen of the prostate 3; FTH1, ferritin heavy chain 1.

Article Snippet: The Kelch-like ECH-associated protein 1 (Keap1) antibody (cat. no. bs-3648R) was purchased from BIOSS.

Techniques: Expressing, Control, Derivative Assay, Membrane

Journal: Molecular Medicine Reports

Article Title: Pathological mechanism of ferroptosis in a rat model of α-naphthyl isothiocyanate-induced chronic cholestasis

doi: 10.3892/mmr.2026.13802

Figure Lengend Snippet: Diagram of the mechanism of ferroptosis. ASCL4, acyl-CoA synthetase long-chain family member 4; COX2, cyclooxygenase 2; DMT1, divalent metal transporter 1; FPN, ferroportin; GSSG, oxidized glutathione; GPX4, glutathione peroxidase 4; GSH, glutathione; HO-1, heme oxygenase 1; Keap1, Kelch-like ECH-associated protein 1; LIP, labile iron pool; LPCAT3, lysophosphatidylcholine acyltransferase 3; NCOA4, nuclear receptor coactivator 4; NOX1, nicotinamide adenine dinucleotide phosphate oxidase 1; NQO1, NAD(P)H quinone dehydrogenase 1; Nrf2, nuclear factor erythroid-2-related factor 2; PL-PUFA-OOH, phospholipid-polyunsaturated fatty acid hydroperoxide; SLC7A11, solute carrier family 7 member 11; Steap3, six-transmembrane epithelial antigen of the prostate 3; TFR1, transferrin receptor 1.

Article Snippet: The Kelch-like ECH-associated protein 1 (Keap1) antibody (cat. no. bs-3648R) was purchased from BIOSS.

Techniques:

Journal: Redox Biology

Article Title: Fibroblast circSamd4 promotes cardiac fibrosis via activating plasminogen activator inhibitor-1

doi: 10.1016/j.redox.2026.104018

Figure Lengend Snippet: CircSamd4 is enriched in cardiac fibroblasts and upregulated in human and mouse fibrotic hearts. (A) Validation of fibrosis and fibrotic gene expression in mouse hearts after TAC. (B) CircRNA array analysis of normal hearts and fibrotic hearts (n = 5). A total of 361 circRNAs were upregulated, whereas 143 were downregulated in fibrotic hearts. (|log 2 FC| ≥ 0.5, P < 0.05). (C) The top 10 overexpressed circRNAs in fibrotic hearts. (D) Differential analysis of the top three expressed circRNAs in normal and heart failure samples of humans (Normal, n = 3; Heart failure, n = 9). (E) qRT-PCR results showing circSamd4 upregulation in human failing hearts (n = 7–14). (F) Expression of the top three circRNAs in primary cardiomyocytes and mouse cardiac fibroblasts stimulated with TGF-β1 (n = 4). (G) Expression of circSamd4 in cardiac fibroblasts derived from the heart of the sham group and TAC group, respectively (n = 5). (H) Representative images and fluorescence intensity statistics of α-SMA in cardiac fibroblasts with or without TGF-β1 treatment for 24h (n = 12). (I) Expression of fibrotic genes Postn , Ccn2 , and circSamd4 in cardiac fibroblasts with or without TGF-β1 treatment (n = 6). (J) Sanger sequencing revealed the head-to-tail junction of circSamd4 . (K) The existence of circSamd4 was confirmed by RT-PCR and gel electrophoresis using convergent and divergent primers. Divergent primers amplified circSamd4 from cDNA but not from genomic DNA. (L) Comparison of the expression of circSamd4 in the cytoplasm and in the nucleus by RT‒qPCR, with Gapdh as an internal reference in the cytoplasm and U6 as an internal reference in the nucleus. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Data A , G was analyzed by Mann–Whitney test, data in D , E , F , H and I was analyzed with Student's t -test.

Article Snippet: Before treatment, the CFs were serum-starved in serum-free DMEM for 4 h. Subsequently, they were treated with 10 ng/mL TGF-β1 (Sino Biological Inc. 80116-RNAH, China) for 24 h to induce activation.

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Expressing, Derivative Assay, Fluorescence, Sequencing, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Amplification, Comparison, MANN-WHITNEY

Journal: Redox Biology

Article Title: Fibroblast circSamd4 promotes cardiac fibrosis via activating plasminogen activator inhibitor-1

doi: 10.1016/j.redox.2026.104018

Figure Lengend Snippet: circSamd4 regulates cardiac fibroblast proliferation and activation. (A) Expression of circSamd4 and its host gene Samd4 after transfection of si- circSamd4 in cardiac fibroblasts (n = 3). (B) The mRNA levels of the fibrotic marker genes Ccn2, Postn, and Acta2 in cardiac fibroblasts are reduced by circSamd4 knockdown (n = 3). (C) circSamd4 knockdown represses TGF-β1-induced α-SMA overexpression in cardiac fibroblasts (n = 3). (D) EdU staining showing circSamd4 knockdown represses TGF-β1-induced proliferation of cardiac fibroblasts (n = 12). (E) circSamd4 knockdown represses TGF-β1-induced deregulated contraction of cardiac fibroblasts (n = 3). (F) circSamd4 knockdown represses TGF-β1-induced migration of cardiac fibroblasts (n = 3). (G) Representative images staining of GFP and RT-qPCR results showing adenovirus-mediated circSamd4 expression in cardiac fibroblasts (n = 3). (H) circSamd4 overexpression promotes the mRNA levels of the fibrotic marker genes Ccn2 , Postn and Acta2 in TGF-β1-treated cardiac fibroblasts (n = 3). (I) circSamd4 overexpression upregulates protein levels of FN1, COL3A1, COL1A2, CTGF, and α-SMA in TGF-β1-treated cardiac fibroblasts. (J) circSamd4 overexpression upregulates protein levels of α-SMA in TGF-β1-treated cardiac fibroblasts (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Data in A and G was analyzed with Student's t -test, data in B , C , D , E , F, H, I and J was analyzed by one-way ANOVA with Bonferroni post hoc multiple comparison test.

Article Snippet: Before treatment, the CFs were serum-starved in serum-free DMEM for 4 h. Subsequently, they were treated with 10 ng/mL TGF-β1 (Sino Biological Inc. 80116-RNAH, China) for 24 h to induce activation.

Techniques: Activation Assay, Expressing, Transfection, Marker, Knockdown, Over Expression, Staining, Migration, Quantitative RT-PCR, Comparison

Journal: Redox Biology

Article Title: Fibroblast circSamd4 promotes cardiac fibrosis via activating plasminogen activator inhibitor-1

doi: 10.1016/j.redox.2026.104018

Figure Lengend Snippet: Serpine1 is a downstream effector of circSamd4 in cardiac fibroblasts. (A) Bulk RNA sequencing was performed to analyze the transcriptome of cardiac fibroblasts (Control, TGF-β1_treated, TGF-β1_treated_plus_ circSamd4 _knockdown) (n = 3). (B) Trend analysis of the differentially expressed genes identified nine key clusters. (C) Heatmap of the top 20 genes in clusters #3 and #4. (D) Gene Ontology (GO) analysis results of genes in clusters #3 and #4 identify the enrichment of the coagulation-regulated pathway (negative regulation of the plasminogen activation). (E) Systemic analysis identifying the PAI-1 coding gene Serpine1 as a circSamd4 downstream effector. The schematic flow chart of the analysis of circSamd4 downstream target genes. Venn diagram showing the overlapping genes identified by the two methods. A total of 6 overlapping genes were identified, and Serpine1 ranks as the top one. (F) Construction of the circRNA-based ceRNA network based on the six overlapping genes identifies miR-1894-3p as a microRNA mediating the effects of circSamd4 on Serpine1 . (G) Single-cell RNA sequencing data (public databases) showed that SERPINE1 is primarily enriched in cardiac fibroblasts in failure human hearts. (H) Correlation analysis between SERPINE1 levels and fibrosis marker genes ( POSTN, CCN2, and FIBRONECTIN ) in human fibrotic hearts. (I) circSamd4 knockdown reduces the mRNA levels of Serpine1 in fibrotic hearts (n = 5–7). (J) circSamd4 knockdown reduces Serpine1 mRNA level in cardiac fibroblasts treated with TGF-β1 (n = 4). (K) circSamd4 knockdown reduces TGF-β1-induced expression of Serpine1- coding PAI-1 in cardiac fibroblasts (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Data in I and J was analyzed by one-way ANOVA with Bonferroni post hoc multiple comparison test.

Article Snippet: Before treatment, the CFs were serum-starved in serum-free DMEM for 4 h. Subsequently, they were treated with 10 ng/mL TGF-β1 (Sino Biological Inc. 80116-RNAH, China) for 24 h to induce activation.

Techniques: RNA Sequencing, Control, Knockdown, Coagulation, Activation Assay, Marker, Expressing, Comparison

Journal: Redox Biology

Article Title: Fibroblast circSamd4 promotes cardiac fibrosis via activating plasminogen activator inhibitor-1

doi: 10.1016/j.redox.2026.104018

Figure Lengend Snippet: CircSamd4 regulates Serpine1 expression by sponging miR-1894-3p. (A) The expression of miR-1894-3p is reduced in fibrotic hearts induced by TAC (n = 5). (B) The expression of miR-1894-3p is reduced in activated fibroblasts induced by TGF-β1 (n = 3). (C) Validation of miR-1894-3p overexpression in cardiac fibroblasts transfected with mimics-miR-1894-3p (n = 3). (D) miR-1894-3p overexpression decreases the protein levels of the fibrotic markers FN1, POSTN, and CTGF in TGF-β1-treated cardiac fibroblasts (n = 3). (E) The expression of miR-1894-3p in cardiac fibroblasts is upregulated by circSamd4 knockdown (n = 3). (F) Prediction of the base pairing of circSamd4 and miR-1894-3p. (G) Verification of miR-1894-3p as a sponge target of circSamd4 via a dual-luciferase reporter assay (n = 4). (H) Relative mRNA expression of Serpine1 in cardiac fibroblasts is reduced by miR-1894-3p overexpression (n = 3). (I) The protein expression of PAI-1 in cardiac fibroblasts is reduced by miR-1894-3p overexpression (n = 3). (J) Prediction of the base pairing of miR-1894-3p and the Serpine1 3′-UTR. (K) Verification of Serpine1 as a target gene of miR-1894-3p via a dual-luciferase reporter assay (n = 4). (L) circSamd4 regulates Serpine1 expression in a miR-1894-3p-depedent manner in cardiac fibroblasts (n = 3). (M) Schematic showing circSamd4 serves as a sponge of miR-1894-3p to release its suppression of Serpine1 expression in cardiac fibroblasts. ns, P > 0.05, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Data in A, B, C, E, G, H, I and K was analyzed with Student's t -test, data in D and L was analyzed by one-way ANOVA with Bonferroni post hoc multiple comparison test.

Article Snippet: Before treatment, the CFs were serum-starved in serum-free DMEM for 4 h. Subsequently, they were treated with 10 ng/mL TGF-β1 (Sino Biological Inc. 80116-RNAH, China) for 24 h to induce activation.

Techniques: Expressing, Biomarker Discovery, Over Expression, Transfection, Knockdown, Luciferase, Reporter Assay, Comparison

Journal: Redox Biology

Article Title: Fibroblast circSamd4 promotes cardiac fibrosis via activating plasminogen activator inhibitor-1

doi: 10.1016/j.redox.2026.104018

Figure Lengend Snippet: circSamd4 regulates cardiac fibroblast activation via the miR-1894-3p- Serpine1 axis. (A–B) The efficiency of siRNA-mediated Serpine1 silencing in cardiac fibroblasts (n = 3). (C) The protein levels of fibrotic markers (COL3A1, COL1A2, and CTGF) were reduced by Serpine1 knockdown in TGF-β1-treated cardiac fibroblasts (n = 3). (D) Serpine1 knockdown reduces TGF-β1-induced α-SMA overexpression in cardiac fibroblasts (n = 3). (E) miR-1894-3p and Serpine1 are involved in circSamd4 regulation of COL3A1, COL1A2, and CTGF protein levels in cardiac fibroblasts (n = 3). (F) miR-1894-3p and Serpine1 are involved in circSamd4 regulation of α-SMA in cardiac fibroblasts (n = 3). (G) miR-1894-3p and Serpine1 are involved in circSamd4 regulation of the migration ability of cardiac fibroblasts (n = 3). (H) miR-1894-3p and Serpine1 are involved in circSamd4 regulation of contraction of cardiac fibroblasts (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Data in A and B was analyzed with Student's t -test, data in C, D, F to H was analyzed by one-way ANOVA with Bonferroni post hoc multiple comparison test.

Article Snippet: Before treatment, the CFs were serum-starved in serum-free DMEM for 4 h. Subsequently, they were treated with 10 ng/mL TGF-β1 (Sino Biological Inc. 80116-RNAH, China) for 24 h to induce activation.

Techniques: Activation Assay, Knockdown, Over Expression, Migration, Comparison

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 promoted the degradation and polyubiquitination of Snail. (A) UBR5 promoted the proteasomal degradation of Snail. HEK293T cells were transfected with Snail-Flag, Snail 6SA-Flag, UBR5-Myc, GFP, or empty vector and treated with DMSO, chloroquine, MG132, or CT99021 as indicated. The expression of Snail and GFP was assessed by western blotting. (B) UBR5 degraded Snail protein in a concentration-dependent manner. HEK293T cells were transfected with Snail-Flag, GFP, or in combination with different concentrations of wild-type and truncated UBR5-Myc for 48 h. Cell lysates were immunoblotted with anti-Snail antibodies. (C) UBR5 promoted K48 polyubiquitinated chain generation of Snail protein. In cellular ubiquitination assays, UBR5-Myc were co-transfected with Snail-Flag plasmids or with HA-Ub-K63 and HA-Ub-K48 plasmids. Western blotting was performed on cell lysates immunoprecipitated with an anti-Flag antibody, followed by the detection of polyubiquitination levels using an anti-Ub antibody. (D) UBR5 accelerated the Snail protein turnover through the HECT domain. HEK293T cells were transfected with corresponding plasmids. Cells were treated with cycloheximide (CHX) and harvested at indicated time points for immunoblotting with anti-Snail or anti-GFP antibody. The graph shows the quantification of Snail protein levels (based on the band intensity from the gels) normalized to those of GFP over the time course. Snail protein expression at the 0 h time point of treatment with CHX was set as 100 %. Experiments were performed in triplicate, and a representative experiment is presented.

Article Snippet: The membranes were probed with primary antibodies, including Flag (Proteintech, Wuhan, China, 66008-4-Ig), Myc (Proteintech, 60003-2-Ig), UBR5 (Proteintech, 66937-1-Ig), Snail (Santa Cruz Biotechnology, Oregon, USA, 166476), phosphorylated Snail (Biodragon, BD-PP0568), Slug (Santa Cruz Biotechnology, 271977), E-cadherin (Proteintech, 20874-1-AP), N-cadherin (BD Transduction Laboratories, Franklin Lakes, USA, 610920), GSK3β (Proteintech, 82061-1-RR), pGSK3β (Proteintech, 67558-1-Ig), green fluorescent protein (GFP; Proteintech, 66002-1-Ig), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Bioss, Woburn, USA, 0978M).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Concentration Assay, Ubiquitin Proteomics, Immunoprecipitation

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 C2768S mutation abrogated the interaction with Snail. (A) His pull-down assays showed the abolished interactions between Snail and the UBR5 C2768S. A schematic representation of the UBR5 wild-type and C2768S mutation. (B) Co-immunoprecipitation assay showed that the interaction between the Snail and the UBR5 C2768S mutation was eliminated. HEK293T cells were transfected with UBR5-Myc, UBR5 C2768S-Myc, and Snail-Flag as indicated. Cell lysates were immunoprecipitated with either anti-Myc or anti-Flag antibodies and immunoblotted with anti-Snail and anti-UBR5 antibodies. (C) UBR5 C2768S abolished the UBR5-mediated degradation of Snail. HEK293T cells were transfected with Snail-Flag, UBR5-Myc, and UBR5 C2768S-Myc as indicated. Cell lysates were subjected to western blotting analysis with anti-Snail and anti-GFP antibodies. (D) UBR5 C2768S did not accelerate Snail protein turnover. HEK293T cells were transfected with Snail-Flag, UBR5-Myc, and UBR5 C2768S-Myc and treated with cycloheximide (CHX) as indicated. Cell lysates were subjected to western blotting analysis with anti-Snail and anti-GFP antibodi.

Article Snippet: The membranes were probed with primary antibodies, including Flag (Proteintech, Wuhan, China, 66008-4-Ig), Myc (Proteintech, 60003-2-Ig), UBR5 (Proteintech, 66937-1-Ig), Snail (Santa Cruz Biotechnology, Oregon, USA, 166476), phosphorylated Snail (Biodragon, BD-PP0568), Slug (Santa Cruz Biotechnology, 271977), E-cadherin (Proteintech, 20874-1-AP), N-cadherin (BD Transduction Laboratories, Franklin Lakes, USA, 610920), GSK3β (Proteintech, 82061-1-RR), pGSK3β (Proteintech, 67558-1-Ig), green fluorescent protein (GFP; Proteintech, 66002-1-Ig), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Bioss, Woburn, USA, 0978M).

Techniques: Mutagenesis, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot