Journal: JHEP Reports

Article Title: Serpine1 as a potential therapeutic target in pyrrolidine alkaloids-induced hepatic sinusoidal obstruction syndrome

doi: 10.1016/j.jhepr.2026.101736

Figure Lengend Snippet: Transcriptomic signatures in PA-HSOS mouse livers. (A) Volcano plot of differential gene expression reveals dysregulation of genes critical to PA-HSOS pathogenesis, with log 2 fold-change and statistical significance plotted to identify transcriptional alterations between PA-HSOS and control livers (n = 6/group). (B) KEGG pathway enrichment analysis identifies dysregulated biological pathways in PA-HSOS livers (n = 6/group). (C) Heatmap of key differentially expressed genes demonstrates distinct transcriptomic signatures distinguishing PA-HSOS from control samples (n = 6/group). (D) Single-cell RNA-seq UMAP clustering delineates distinct hepatic cell populations and their transcriptional heterogeneity in PA-HSOS vs. control livers (n = 3/group). (E) Violin plots of Serpine1 expression in LSECs (n = 3/group). (F) Ridge plot visualization further characterizes the dynamic range of Serpine1 expression in LSECs (n = 3/group). All data represent means ± SEM. LSEC, liver sinusoidal endothelial cell; PA-HSOS, pyrrolizidine alkaloid–induced hepatic sinusoidal obstruction syndrome; RNA-seq, RNA sequencing; UMAP, uniform manifold approximation and projection.

Article Snippet: Following the 28-day establishment of the PA-HSOS model, C57BL/6 mice underwent treatment with the Serpine1 inhibitor TM5441 (Cat# HY-101761, MCE).

Techniques: Gene Expression, Control, Single Cell, RNA Sequencing, Expressing

Journal: JHEP Reports

Article Title: Serpine1 as a potential therapeutic target in pyrrolidine alkaloids-induced hepatic sinusoidal obstruction syndrome

doi: 10.1016/j.jhepr.2026.101736

Figure Lengend Snippet: The Serpine1/p53 pathway activation and associated pathologies in vivo . (A) qPCR analysis demonstrates upregulation of Serpine1 and p5 3 mRNA in mouse LSECs from PA-HSOS mice (n = 6/group). (B) Western blot analysis of mouse LSECs confirms elevated Serpine1 protein alongside increased p53, p21, and p16 expression. (C) Flow cytometry-based cell cycle analysis reveals G1/S phase arrest in PA-HSOS LSECs (n = 6/group). (D) qPCR quantification of senescence-associated molecules in mouse LSECs shows increased expression of key senescence markers (n = 6/group). (E) Immunofluorescence intensity of p16 in ERG-positive cells in mouse livers localizes senescent endothelial cells within hepatic sinusoids (n = 6/group). Scale bars, 50 μm. (F) TUNEL staining reveals enhanced endothelial cell apoptosis in PA-HSOS livers compared to controls (n = 6/group). Scale bars, 200 μm (or 50 μm for enlarged regions). (G) Flow cytometry of mouse LSECs quantifies apoptotic populations (n = 6/group). (H) Western blot analysis of Bax and cleaved caspase-3 in mouse LSECs confirms activation of the intrinsic apoptotic pathway. (I) In vivo endothelial permeability assay using tail-vein injection of 40 kDa FITC-dextran demonstrates compromised sinusoidal barrier function in PA-HSOS mice, with quantified fluorescence intensity reflecting enhanced vascular leakage into hepatic tissue (n = 6/group). Scale bars, 100 μm (or 50 μm for enlarged regions). (J) Western blot of eNOS and VE-cadherin in mouse LSECs reveals reduced expression of these vascular integrity markers. (K) Serum Serpine1 levels are significantly elevated in PA-HSOS mice and humans compared to respective controls (mice: n = 6/group; humans: n = 16/group). All data represent means ± SEM. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by Mann-Whitney U test.

Article Snippet: Following the 28-day establishment of the PA-HSOS model, C57BL/6 mice underwent treatment with the Serpine1 inhibitor TM5441 (Cat# HY-101761, MCE).

Techniques: Activation Assay, In Vivo, Western Blot, Expressing, Flow Cytometry, Cell Cycle Assay, Immunofluorescence, TUNEL Assay, Staining, Permeability, Injection, Fluorescence, MANN-WHITNEY

Journal: JHEP Reports

Article Title: Serpine1 as a potential therapeutic target in pyrrolidine alkaloids-induced hepatic sinusoidal obstruction syndrome

doi: 10.1016/j.jhepr.2026.101736

Figure Lengend Snippet: PA-induced endothelial dysfunction and senescence in a hepatocyte-LSEC co-culture system. (A) Schematic representation of the hepatocyte-LSEC co-culture system, with hepatocytes cultured in the upper Transwell compartment and LSECs in the lower chamber, enabling paracrine signaling and physiologically relevant PA metabolite exposure. (B) PA treatment impairs LSEC morphology and viability in co-culture, as demonstrated by phase-contrast microscopy and quantified by CCK-8 assay (n = 3 independent experiments). Scale bars, 100 μm. (C) qPCR analysis reveals transcriptional upregulation of Serpine1 and p53 alongside their downstream effectors p21 and p16 in PA-exposed LSECs (n = 3 independent experiments). (D) Western blot analysis demonstrates corresponding increases in Serpine1, p53, p21, and p16 protein levels in LSECs following PA treatment. (E) SA-β-Gal staining quantifies senescent LSECs following PA exposure, providing morphological evidence of endothelial senescence induction and confirming functional engagement of the senescence program (n = 3 independent experiments). (F) qPCR profiling of inflammatory cytokines and SASP genes demonstrates coordinated upregulation of pro-inflammatory mediators and senescence markers in PA-treated LSECs (n = 3 independent experiments). (G) Western blot analysis of Serpine1, p53, p21, and p16 in PA-exposed LSECs treated with si-Serpine1 demonstrates that Serpine1 knockdown attenuates p53-pathway activation and senescence markers. All data represent means ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by Mann-Whitney U test. n = 3/group (B-G). LSEC, liver sinusoidal endothelial cell; PA, pyrrolizidine alkaloid; qPCR, quantitative PCR; SA-β-Gal, senescence-associated β-galactosidase; SASP, senescence-associated secretory phenotype; si, small-interfering.

Article Snippet: Following the 28-day establishment of the PA-HSOS model, C57BL/6 mice underwent treatment with the Serpine1 inhibitor TM5441 (Cat# HY-101761, MCE).

Techniques: Co-Culture Assay, Cell Culture, Microscopy, CCK-8 Assay, Western Blot, Staining, Functional Assay, Knockdown, Activation Assay, MANN-WHITNEY, Real-time Polymerase Chain Reaction

Journal: JHEP Reports

Article Title: Serpine1 as a potential therapeutic target in pyrrolidine alkaloids-induced hepatic sinusoidal obstruction syndrome

doi: 10.1016/j.jhepr.2026.101736

Figure Lengend Snippet: Serpine1 mediates LSEC dysfunction through cell cycle arrest, fenestration loss, and apoptosis in PA-HSOS. (A) Representative flow cytometry histograms and quantification of propidium iodide-stained LSECs reveal G1/S arrest in PA-exposed LSECs, which are reversed by Serpine1 knockdown. (B) Scanning electron microscopy visualizes LSEC fenestrations in control and PA-treated conditions, with yellow arrows highlighting characteristic pores. Fenestration density (number per unit area) is significantly reduced in PA-HSOS LSECs but restored upon Serpine1 inhibition. Scale bar, 5 μm. (C) Immunofluorescence shows disrupted VE-cadherin organization in PA-treated LSECs, with reduced VE-cadherin expression partially recovered by Serpine1 knockdown. Scale bar, 100 μm. (D) In vitro permeability assay using 40 kDa FITC-dextran leakage quantifies barrier dysfunction in PA-exposed LSECs, demonstrating enhanced paracellular flux that reflects compromised endothelial integrity and is attenuated by Serpine1 knockdown. (E) Western blot analysis of endothelial function-related proteins (including eNOS and VE-cadherin) and apoptotic markers, including pro-apoptotic factors (cleaved caspase-3 and Bax) reveals downregulated endothelial function and activation of the apoptotic cascade in PA-treated LSECs, which is suppressed by Serpine1 inhibition. (F) Representative flow cytometry analysis delineates that PA-induced LSEC apoptosis is significantly reduced by Serpine1 knockdown. Data represent means ± SEM (n = 3/group). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by Mann-Whitney U test. n = 3/group independent experiments. LSEC, liver sinusoidal endothelial cell; PA, pyrrolizidine alkaloid; PA-HSOS, pyrrolizidine alkaloid–induced hepatic sinusoidal obstruction syndrome.

Article Snippet: Following the 28-day establishment of the PA-HSOS model, C57BL/6 mice underwent treatment with the Serpine1 inhibitor TM5441 (Cat# HY-101761, MCE).

Techniques: Flow Cytometry, Staining, Knockdown, Electron Microscopy, Control, Inhibition, Immunofluorescence, Expressing, In Vitro, Permeability, Western Blot, Activation Assay, MANN-WHITNEY

Journal: JHEP Reports

Article Title: Serpine1 as a potential therapeutic target in pyrrolidine alkaloids-induced hepatic sinusoidal obstruction syndrome

doi: 10.1016/j.jhepr.2026.101736

Figure Lengend Snippet: Molecular interaction between Serpine1 and p53 in PA-HSOS pathogenesis. (A) Western blot analysis of p53 and Serpine1 in LSECs treated with si-Serpine1 or si-p53, showing reciprocal regulation of p53 and Serpine1 protein levels under PA exposure. (B) qPCR analysis showing that Serpine1 or p53 knockdown does not significantly alter p53 or Serpine1 mRNA levels in PA-treated LSECs. (C) Cycloheximide (CHX) chase assay combined with autophagy inhibitor (3-MA) or proteasome inhibitor (MG-132) demonstrating that Serpine1 stabilizes p53 protein by inhibiting proteasomal degradation. (D) Co-immunoprecipitation analysis of human liver samples showing enhanced interaction between Serpine1 and p53 in PA-HSOS compared with control tissue. (E) Immunofluorescence staining demonstrating subcellular colocalization of Serpine1 and p53 in LSECs under PA-HSOS conditions. Scale bar, 5 µm. (F) Co-immunoprecipitation analysis showing that Serpine1 interferes with the interaction between p53 and MDM2. (G) Ubiquitination assay demonstrating increased p53 polyubiquitination following Serpine1 knockdown, indicating enhanced p53 degradation. (H) Western blot analysis showing that Serpine1 knockdown promotes MDM2-dependent p53 degradation, whereas intact Serpine1 expression stabilizes p53 protein levels. Data represent means ± SEM (n = 3/group). LSEC, liver sinusoidal endothelial cell; PA-HSOS, pyrrolizidine alkaloid–induced hepatic sinusoidal obstruction syndrome; qPCR, quantitative PCR; si, small-interfering.

Article Snippet: Following the 28-day establishment of the PA-HSOS model, C57BL/6 mice underwent treatment with the Serpine1 inhibitor TM5441 (Cat# HY-101761, MCE).

Techniques: Western Blot, Knockdown, Immunoprecipitation, Control, Immunofluorescence, Staining, Ubiquitin Proteomics, Expressing, Real-time Polymerase Chain Reaction

Journal: JHEP Reports

Article Title: Serpine1 as a potential therapeutic target in pyrrolidine alkaloids-induced hepatic sinusoidal obstruction syndrome

doi: 10.1016/j.jhepr.2026.101736

Figure Lengend Snippet: Therapeutic efficacy of TM5441 in ameliorating PA-HSOS. (A) Schematic overview of the experimental design for PA-HSOS mouse model treatment with TM5441, a selective p53 activator. (B) Body weight trajectories demonstrate that TM5441 treatment significantly attenuates PA-induced weight loss. (C) Hemodynamic analysis reveals that TM5441 restores abnormal hemodynamic parameters, including PP, MAP, and PBF. (D) Comprehensive biochemical assessment demonstrates that TM5441 treatment ameliorates liver dysfunction (ALT, AST, ALP), coagulation abnormalities (PT, aPTT, D-dimer), systemic inflammation (IL-1β, TNF-α), and hematological dysregulation (platelets, RBC, WBC). (E) Histopathological analysis including H&E, Masson trichrome, IL-1β immunohistochemistry, and DAPI/CD31/VE-cadherin immunofluorescence demonstrates that TM5441 reduces sinusoidal RBC accumulation, hepatic fibrosis, inflammatory infiltration, and endothelial dysfunction. Black arrows indicate RBC accumulation within hepatic sinusoids. Scale bars, 200 μm (or 50 μm for enlarged regions). (F) FITC-dextran permeability assay with immunofluorescence visualization demonstrates that TM5441 restores sinusoidal barrier integrity and reduces vascular leakage. Scale bars, 100 μm (or 50 μm for enlarged regions). (G) qPCR profiling reveals that TM5441 suppresses inflammatory cytokine expression and reduces senescence-associated gene upregulation in liver tissue. (H) Western blot analysis of LSEC function and apoptotic markers shows that TM5441 reduces pro-apoptotic protein levels and restores anti-apoptotic factors. (I) Flow cytometry demonstrates that TM5441 significantly reduces apoptosis rates in hepatic endothelial cells. (J) Western blot of the Serpine1-p53 pathway reveals that TM5441 downregulates Serpine1 and p53 protein levels alongside reduced expression of downstream senescence effectors, including p21 and p16. (K) Immunofluorescence of ERG and p16 in hepatic tissue reveals reduced coexpression in TM5441-treated mice. Scale bar, 50 μm. (L) Flow cytometry-based cell cycle analysis demonstrates that TM5441 reverses PA-induced G1/S phase arrest in hepatic endothelial cells. Data represent means ± SEM (n = 6/group). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by Mann-Whitney U test. ALP, alkaline phosphatase; ALT, alanine aminotransferase; aPTT, activated partial thromboplastin time; AST, aspartate aminotransferase; LSEC, liver sinusoidal endothelial cell; MAP, mean arterial pressure; PA-HSOS, pyrrolizidine alkaloid–induced hepatic sinusoidal obstruction syndrome; PBF, portal blood flow; PP, portal pressure; PT, prothrombin time; qPCR, quantitative PCR; RBC, red blood cell; WBC, white blood cell.

Article Snippet: Following the 28-day establishment of the PA-HSOS model, C57BL/6 mice underwent treatment with the Serpine1 inhibitor TM5441 (Cat# HY-101761, MCE).

Techniques: Drug discovery, Coagulation, Immunohistochemistry, Immunofluorescence, FITC-Dextran Permeability Assay, Expressing, Western Blot, Flow Cytometry, Cell Cycle Assay, MANN-WHITNEY, Real-time Polymerase Chain Reaction

Journal: medRxiv

Article Title: Assessing the clinical significance of a novel rare variant in Loeys-Dietz Syndrome by combining AI-driven modelling and cell biology

doi: 10.64898/2026.03.30.26349510

Figure Lengend Snippet: TGFBR2 E431K disrupts the canonical TGF-β signalling pathway in vitro. HEK293T cells were transiently transfected with 0.5 μg of pcDNA3.1+ empty vector, WT TGFBR2, or TGFBR2 E431K for 48 h and stimulated with TGF-β1 (10 ng/mL). Western blotting was used to assess (a) TGFBR2 abundance and SMAD pathway activation, including (b) whole-cell pSMAD2 and total SMAD2/3 and (c) nuclear pSMAD2 at 1 h post-stimulation, as well as (d) pSMAD2 levels at 1, 6, and 9 h post-stimulation. Downstream transcriptional responses ( SERPINE1 , COL1A1 ) were quantified by RT–qPCR. All experiments were performed in triplicate; Data are shown as mean ± Standard error of the mean (SEM), with statistical significance of *p < 0.05 , **p < 0.01 , ***p < 0.001 , and ****p < 0.0001

Article Snippet: Each reaction was carried out in triplicate in a 20 μL volume containing TaqManTM Universal PCR Master Mix, cDNA template, and gene-specific TaqManTM probes (TGFBR2 Hs00234253_m1 COL1A1 Hs00164004_m1, SERPINE-1 Hs01126607_g1.

Techniques: In Vitro, Transfection, Plasmid Preparation, Western Blot, Activation Assay, Quantitative RT-PCR

Journal: International Journal of Oncology

Article Title: Diverse roles of SERPINE1 in regulating cellular proliferation and invasion

doi: 10.3892/ijo.2026.5871

Figure Lengend Snippet: Prognostic significance and cellular functional states of SERPINE1. (A) Heatmap of survival (GEPIA2) showing the HRs for SERPINE1 expression across type of cancers (expression data and clinical survival data are sourced from The Cancer Genome Atlas projects. The cohort was split into two groups based on the expression level of SERPINE1. GEPIA2 employs a quartile-based cut-off: tumors with SERPINE1 expression levels at or above the 75th percentile are classified into the 'high expression' group, and those with SERPINE1 expression at or below the 25th percentile are classified into the 'low expression' group. This method minimizes the effects of outliers. Red and blue indicate higher and lower risks, respectively; highlighted boxes denote significant outcomes (P<0.05). (B) Kaplan-Meier curves for the significance of the effect of SERPINE1 on overall survival. The statistical significance of the difference between survival curves was computed using the log-rank (Mantel-Cox) test. The analysis outputs HR with its confidence interval and the log-rank P-value, all of which are annotated in the figure. (C) SERPINE1 expression in BRCA, LGG, and SKCM tumors compared with normal tissues (GEPIA2). For BRCA, there were 1,085 tumor samples and 291 normal samples; for LGG, 518 tumor samples and 207 normal samples; and for SKCM, 461 tumor samples and 558 normal samples. (D) Average correlation of SERPINE1 expression with functional states in cancers (CancerSEA database). Correlations with an absolute Spearman's ρ>0.3 and a Benjamini and Hochberg adjusted P-value (false discovery rate) <0.05 were considered significant. * P<0.05. SERPINE1, serine protease inhibitor clade e member 1; GEPIA2, gene expression profiling interactive analysis 2; HR, hazard ratio; BRCA, breast cancer; LGG, low-grade glioma; SKCM, skin cutaneous melanoma.

Article Snippet: The concentration of SERPINE1 in the cell culture supernatant was measured using a human SERPINE1 ELISA Kit (cat. no. EH0538; Wuhan Fine Biotech Co., Ltd.).

Techniques: Functional Assay, Expressing, Protease Inhibitor, Gene Expression

Journal: International Journal of Oncology

Article Title: Diverse roles of SERPINE1 in regulating cellular proliferation and invasion

doi: 10.3892/ijo.2026.5871

Figure Lengend Snippet: Diverse effects of SERPINE1 knockdown on cell proliferation. (A) Western blotting showing SERPINE1 protein levels in 231, H4, and C918 cells following transfection with the shSE1 and shc with GAPDH serving as the loading control. Band intensities were measured using ImageJ software and are presented as ratios. These ratios were calculated as (target protein/GAPDH levels) in the experimental group divided by those in the control group. Data represent mean ± SDs of three independent experiments. (B) Colony formation ability of shSE1 and control (shc) cells. Statistical significance was determined using a two-sided Student's t test. The data are presented as the means ± SDs of three independent experiments. (C-D) Xenograft tumors derived from (C) 231 and (D) C918 cells with shSE1 and shc cells; the tumor growth and weight were compared between shSE1 and shc groups. Statistical significance was determined using two-way ANOVA followed by Šídák's multiple comparisons test (tumor growth) and a two-sided Student's t test (tumor weight). The data are presented as the means ± SDs. n=5. (E-F) Lung metastatic foci in nude mice were stained with H&E and GFP (scale bar, 100 μ m). The fractions (numerator/denominator) adjacent to the images represent the lung metastatic focus rate (defined as the number of mice with lung metastases per total number of injected mice). The lung metastatic focus rate was: 4 of 6 for 231-shSE1 versus 5 of 6 for 231-shc; and 6 of 6 for both C918-shSE1 and C918-shc. Statistical significance was determined using a two-sided Student's t test. The data are presented as the means ± SDs. n=6. *** P<0.001, ** P<0.01. SERPINE1, serine protease inhibitor clade e member 1; shRNA, short hairpin RNA; shSE1, shRNA targeting SERPINE1; shc, shRNA scrambled control; H&E, hematoxylin and eosin staining; GFP, green fluorescent protein.

Article Snippet: The concentration of SERPINE1 in the cell culture supernatant was measured using a human SERPINE1 ELISA Kit (cat. no. EH0538; Wuhan Fine Biotech Co., Ltd.).

Techniques: Knockdown, Western Blot, Transfection, Control, Software, Derivative Assay, Staining, Injection, Protease Inhibitor, shRNA

Journal: International Journal of Oncology

Article Title: Diverse roles of SERPINE1 in regulating cellular proliferation and invasion

doi: 10.3892/ijo.2026.5871

Figure Lengend Snippet: SERPINE1-mediated cell cycle regulation. (A) The GO enrichment analysis of DEGs obtained from RNA-seq revealed significant changes in biological processes after SERPINE1 knockdown between shSE1 and shc groups across three cell lines. RNA-seq was performed using one biological replicate per group. (B) Heatmap of DEGs associated with the highlighted processes in (A). (C) Western blotting was performed on nuclear and cytoplasmic extracts to detect MCM3 and p-MCM3 levels (histone H3 and GAPDH were used as controls). The relative levels of p-MCM3 and MCM3 were normalized to the expression of histone H3 and GAPDH, respectively. The relative p-MCM3/MCM3 ratio was subsequently calculated. Data represent mean ± SDs of three independent experiments. (D) Flow cytometry of the cell cycle phase distribution (G 0 /G 1 , S, and G 2 /M phases). Statistical significance was determined using a two-sided Student's t test. The data are presented as the means ± SDs of three independent experiments. (E) Scatter plot of 89 cycle regulators from the protein array, highlighting SMAD3 and TP53. Blue indicates downregulated proteins (fold change ≥1.2); red indicates upregulated proteins (fold change ≤0.83); and grey indicates no change in the protein level. n=4 (F) RNA-seq data showing changes in the indicated gene expression between shSE1 and shc groups across three cell lines. RNA-seq was performed using one biological replicate per group. (G) Western blotting of the indicated proteins in C918 cells (shSE1 vs. shc). The ratios indicate the relative changes in the expression levels of the indicated proteins. Data represent mean ± SDs of three independent experiments. * P<0.05; ns, not significant. SERPINE1, serine protease inhibitor clade e member 1; GO, Gene Ontology; DEGs, differentially expressed genes; RNA-seq, RNA sequencing; shRNA, short hairpin RNA; shSE1, shRNA targeting SERPINE1; shc, shRNA scrambled control; FDR, false discovery rate; p-, phosphorylated.

Article Snippet: The concentration of SERPINE1 in the cell culture supernatant was measured using a human SERPINE1 ELISA Kit (cat. no. EH0538; Wuhan Fine Biotech Co., Ltd.).

Techniques: RNA Sequencing, Knockdown, Western Blot, Expressing, Flow Cytometry, Protein Array, Gene Expression, Protease Inhibitor, shRNA, Control

Journal: International Journal of Oncology

Article Title: Diverse roles of SERPINE1 in regulating cellular proliferation and invasion

doi: 10.3892/ijo.2026.5871

Figure Lengend Snippet: SERPINE1-mediated modulation of the ERK/p38 ratio and anoikis. (A) Upper panel: western blotting showing ERK, p-ERK, p38, and p-p38 levels. Quantitative data showed the p-ERK/ERK and p-p38/p38 ratios in SERPINE1 knockdown cells compared with control cells. Lower panel: western blotting analysis of p-AKT, AKT, p-JNK and JNK (normalized to total protein) levels in the shSE1 and shc groups. The numbers beneath the bands represent the ratios of the expression levels of the indicated proteins. Data represent mean ± SDs of three independent experiments. (B) PLAUR and HSP90AA1 mRNA and protein levels in shSE1 and shc cells analyzed using RNA-seq (left panel) and western blotting (right panel). The numbers beneath the bands represent the ratios of the expression levels of the indicated proteins and represent mean ± SDs of three independent experiments. RNA-seq was performed using one biological replicate per group. (C-D) Upper panel: (C) 231-shSE1 cells were infected with lentivirus containing PLAUR cDNA (ex-PLAUR) or the corresponding empty vector control (vec). (D) H4-shSE1 cells were transfected with siRNA targeting HSP90AA1 (si-HSP90AA1) or siNC. The expression levels of uPAR and HSP90α, as well as the activity of ERK and p38, were detected by western blotting, with GAPDH serving as the loading control. Data represent mean ± SDs of three independent experiments. Lower panel: Cell proliferation was assessed by CCK-8 assay in 231-shSE1 cells infected with ex-PLAUR or vec, and in H4-shSE1 cells transfected with si-HSP90AA1 or siNC. Statistical significance was determined using two-way ANOVA followed by Šídák's multiple comparisons test. The data are presented as the means ± SDs. n=6. (E) Morphology of shSE1 and shc cells cultured under suspension condition. (scale bar, 100 μ m). (F) Apoptotic cells cultured under suspension condition were analyzed by flow cytometry following Annexin V-PE/7AAD staining (72 h of suspension culture). Statistical significance was determined using a two-sided Student's t test. The data are presented as the means ± SDs of three independent experiments. ** P<0.01 *** P<0.001, ns, not significant. SERPINE1, serine protease inhibitor clade e member 1; p-, phosphorylated; shRNA, short hairpin RNA; shSE1, shRNA targeting SERPINE1; shc, shRNA scrambled control; PLAUR, plasminogen activator, urokinase receptor; HSP90AA1, heat shock protein 90 alpha family class a member 1; RNA-seq, RNA sequencing; vec, empty vector control; siRNA, short interfering RNA; siNC, negative control siRNA; uPAR, urokinase-type plasminogen activator receptor; HSP90α, heat shock protein 90-alpha.

Article Snippet: The concentration of SERPINE1 in the cell culture supernatant was measured using a human SERPINE1 ELISA Kit (cat. no. EH0538; Wuhan Fine Biotech Co., Ltd.).

Techniques: Western Blot, Knockdown, Control, Expressing, RNA Sequencing, Infection, Plasmid Preparation, Transfection, Activity Assay, CCK-8 Assay, Cell Culture, Suspension, Flow Cytometry, Staining, Protease Inhibitor, shRNA, Small Interfering RNA, Negative Control

Journal: International Journal of Oncology

Article Title: Diverse roles of SERPINE1 in regulating cellular proliferation and invasion

doi: 10.3892/ijo.2026.5871

Figure Lengend Snippet: Variable effects of SERPINE1 on cell migration and invasion. (A) KEGG enrichment analysis via GSEA comparing high and low SERPINE1 expression in BRCA, LGG, and SKCM (TCGA dataset). Representative images and quantification of Transwell assays of the migration (B) of shSE1 and shc cells. Representative images and quantification of Transwell invasion assays through (C) Matrigel and (D) collagen type I. Cells from three random fields in triplicate wells were counted. Scale bar, 100 μ m. Statistical significance was determined using a two-sided Student's t test. The data are presented as the means ± SDs of three independent experiments. ** P<0.01, * P<0.05. SERPINE1, serine protease inhibitor clade e member 1; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, gene set enrichment analysis; BRCA, breast cancer; LGG, low-grade glioma; SKCM, skin cutaneous melanoma; TCGA, The Cancer Genome Atlas; NES, normalized enrichment score; FDR, false discovery rate.

Article Snippet: The concentration of SERPINE1 in the cell culture supernatant was measured using a human SERPINE1 ELISA Kit (cat. no. EH0538; Wuhan Fine Biotech Co., Ltd.).

Techniques: Migration, Expressing, Protease Inhibitor

Journal: International Journal of Oncology

Article Title: Diverse roles of SERPINE1 in regulating cellular proliferation and invasion

doi: 10.3892/ijo.2026.5871

Figure Lengend Snippet: SERPINE1 regulates MMP activity. (A) MMP and TIMP levels in the supernatants of shSE1 and shc cells after 24 h of incubation and 10-fold concentration. Statistical significance was determined using a two-sided Student's t test. The data are presented as the means ± SDs. n=4. (B) Quantification of active MMP-1 and MMP-13 levels in cell lysates using fluorescence ELISA. Statistical significance was determined using two-way ANOVA followed by Šídák's multiple comparisons test. The data are presented as the means ± SDs of three independent experiments. (C) Western blotting showing the levels of the indicated proteins in H4-shSE1 cells at 72 h after transfection with the si-HSP90AA1 or siNC. The numbers beneath the bands represent the ratios of the expression levels of the indicated proteins and represent mean ± SDs of three independent experiments. (D) Representative images and quantification of Transwell invasion assays through Matrigel and collagen type I in H4-shSE1 cells transfected with the si-HSP90AA1 or siNC (scale bar, 100 μ m). Statistical significance was determined using a two-sided Student's t-test. The data are presented as the means ± SDs of three independent experiments. (E) Diagram showing the mechanisms underlying the effects of SERPINE1 on cancer proliferation and invasion. SERPINE1 knockdown influences cell proliferation and invasion through distinct signaling pathways. With respect to proliferation, SERPINE1 knockdown reduces TGF-β levels, and this reduction alters the activity of SMAD3, p53, and MCM3 to promote cell cycle progression. SERPINE1 knockdown interferes with the uPAR-mediated balance of the ERK/p38 ratio; it may also affect this ratio by modulating HSP90α expression and p38 activity, which suppress cell proliferation. In terms of invasion, SERPINE1 downregulation increases MMP-1 levels via the HSP90α-p38 pathway, thereby promoting cellular invasion. *** P<0.001, ** P<0.01, ns, not significant. SERPINE1, serine protease inhibitor clade e member 1; MMP, matrix metalloproteinases; TIMP, tissue inhibitors of metal proteases; shRNA, short hairpin RNA; shSE1, shRNA targeting SERPINE1; si, short interfering RNA; siHSP90AA1, short interfering heat shock protein 90 alpha family class a member 1; NC, negative control; uPAR, urokinase-type plasminogen activator receptor; p-, phosphorylated; MCM3, minichromosome maintenance complex component 3; HSP90α, heat shock protein 90-alpha.

Article Snippet: The concentration of SERPINE1 in the cell culture supernatant was measured using a human SERPINE1 ELISA Kit (cat. no. EH0538; Wuhan Fine Biotech Co., Ltd.).

Techniques: Activity Assay, Incubation, Concentration Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Expressing, Knockdown, Protein-Protein interactions, Protease Inhibitor, shRNA, Small Interfering RNA, Negative Control

Journal: International Journal of Oncology

Article Title: Diverse roles of SERPINE1 in regulating cellular proliferation and invasion

doi: 10.3892/ijo.2026.5871

Figure Lengend Snippet: Prognostic significance and cellular functional states of SERPINE1. (A) Heatmap of survival (GEPIA2) showing the HRs for SERPINE1 expression across type of cancers (expression data and clinical survival data are sourced from The Cancer Genome Atlas projects. The cohort was split into two groups based on the expression level of SERPINE1. GEPIA2 employs a quartile-based cut-off: tumors with SERPINE1 expression levels at or above the 75th percentile are classified into the 'high expression' group, and those with SERPINE1 expression at or below the 25th percentile are classified into the 'low expression' group. This method minimizes the effects of outliers. Red and blue indicate higher and lower risks, respectively; highlighted boxes denote significant outcomes (P<0.05). (B) Kaplan-Meier curves for the significance of the effect of SERPINE1 on overall survival. The statistical significance of the difference between survival curves was computed using the log-rank (Mantel-Cox) test. The analysis outputs HR with its confidence interval and the log-rank P-value, all of which are annotated in the figure. (C) SERPINE1 expression in BRCA, LGG, and SKCM tumors compared with normal tissues (GEPIA2). For BRCA, there were 1,085 tumor samples and 291 normal samples; for LGG, 518 tumor samples and 207 normal samples; and for SKCM, 461 tumor samples and 558 normal samples. (D) Average correlation of SERPINE1 expression with functional states in cancers (CancerSEA database). Correlations with an absolute Spearman's ρ>0.3 and a Benjamini and Hochberg adjusted P-value (false discovery rate) <0.05 were considered significant. * P<0.05. SERPINE1, serine protease inhibitor clade e member 1; GEPIA2, gene expression profiling interactive analysis 2; HR, hazard ratio; BRCA, breast cancer; LGG, low-grade glioma; SKCM, skin cutaneous melanoma.

Article Snippet: Following the blocking of nonspecific binding sites using 5% skimmed milk (cat. no. P0216; Beyotime Biotechnology) or bovine serum albumin (cat. no. NGP0028A; Beyotime Biotechnology) for 1 h at room temperature, the membranes were incubated with primary antibodies (incubation overnight at 4°C) against SERPINE1 (cat. no. 13801-1-AP; Proteintech Group, Inc), GAPDH (cat. no. sc-25778; Santa Cruz Biotechnology, Inc.), MCM3 (cat. no. PA5-79646; Thermo Fisher Scientific, Inc.), phosphorylated (p-)MCM3 (Ser112; cat. no. TA2362; Abmart Pharmaceutical Technology Co., Ltd.), ERK (cat. no. sc-514302; Santa Cruz Biotechnology, Inc.), uPAR (cat. no. ab10379; Abcam), Histone H3 (cat. no. 4499), p-p53 (Ser15; cat. no. 9284), p53 (cat. no. 2524), p-SMAD3 (Ser423/425; cat. no. 9520), SMAD3 (cat. no. 9523), p-Rb (Ser807/811; cat. no. 8516), Rb (cat. no. 9309), CyclinD1 (cat. no. 55506), CyclinE1 (cat. no. 20808), p21 (cat. no. 2947), p-p38 (cat. no. 4511), p38 (cat. no. 8690), p-ERK (cat. no. 4370), p-JNK (cat. no. 9255), JNK (cat. no. 9252), p-AKT (Ser473; cat. no. 4060), AKT (cat. no. 9272), HSP90α (cat. no. 4877) and MMP-1 (cat. no. 54376) were from Cell Signaling Technology, Inc.) and subsequently incubated with the corresponding secondary antibodies (dilution 1:4,000) for 2 h at room temperature.

Techniques: Functional Assay, Expressing, Protease Inhibitor, Gene Expression

Journal: International Journal of Oncology

Article Title: Diverse roles of SERPINE1 in regulating cellular proliferation and invasion

doi: 10.3892/ijo.2026.5871

Figure Lengend Snippet: Diverse effects of SERPINE1 knockdown on cell proliferation. (A) Western blotting showing SERPINE1 protein levels in 231, H4, and C918 cells following transfection with the shSE1 and shc with GAPDH serving as the loading control. Band intensities were measured using ImageJ software and are presented as ratios. These ratios were calculated as (target protein/GAPDH levels) in the experimental group divided by those in the control group. Data represent mean ± SDs of three independent experiments. (B) Colony formation ability of shSE1 and control (shc) cells. Statistical significance was determined using a two-sided Student's t test. The data are presented as the means ± SDs of three independent experiments. (C-D) Xenograft tumors derived from (C) 231 and (D) C918 cells with shSE1 and shc cells; the tumor growth and weight were compared between shSE1 and shc groups. Statistical significance was determined using two-way ANOVA followed by Šídák's multiple comparisons test (tumor growth) and a two-sided Student's t test (tumor weight). The data are presented as the means ± SDs. n=5. (E-F) Lung metastatic foci in nude mice were stained with H&E and GFP (scale bar, 100 μ m). The fractions (numerator/denominator) adjacent to the images represent the lung metastatic focus rate (defined as the number of mice with lung metastases per total number of injected mice). The lung metastatic focus rate was: 4 of 6 for 231-shSE1 versus 5 of 6 for 231-shc; and 6 of 6 for both C918-shSE1 and C918-shc. Statistical significance was determined using a two-sided Student's t test. The data are presented as the means ± SDs. n=6. *** P<0.001, ** P<0.01. SERPINE1, serine protease inhibitor clade e member 1; shRNA, short hairpin RNA; shSE1, shRNA targeting SERPINE1; shc, shRNA scrambled control; H&E, hematoxylin and eosin staining; GFP, green fluorescent protein.

Article Snippet: Following the blocking of nonspecific binding sites using 5% skimmed milk (cat. no. P0216; Beyotime Biotechnology) or bovine serum albumin (cat. no. NGP0028A; Beyotime Biotechnology) for 1 h at room temperature, the membranes were incubated with primary antibodies (incubation overnight at 4°C) against SERPINE1 (cat. no. 13801-1-AP; Proteintech Group, Inc), GAPDH (cat. no. sc-25778; Santa Cruz Biotechnology, Inc.), MCM3 (cat. no. PA5-79646; Thermo Fisher Scientific, Inc.), phosphorylated (p-)MCM3 (Ser112; cat. no. TA2362; Abmart Pharmaceutical Technology Co., Ltd.), ERK (cat. no. sc-514302; Santa Cruz Biotechnology, Inc.), uPAR (cat. no. ab10379; Abcam), Histone H3 (cat. no. 4499), p-p53 (Ser15; cat. no. 9284), p53 (cat. no. 2524), p-SMAD3 (Ser423/425; cat. no. 9520), SMAD3 (cat. no. 9523), p-Rb (Ser807/811; cat. no. 8516), Rb (cat. no. 9309), CyclinD1 (cat. no. 55506), CyclinE1 (cat. no. 20808), p21 (cat. no. 2947), p-p38 (cat. no. 4511), p38 (cat. no. 8690), p-ERK (cat. no. 4370), p-JNK (cat. no. 9255), JNK (cat. no. 9252), p-AKT (Ser473; cat. no. 4060), AKT (cat. no. 9272), HSP90α (cat. no. 4877) and MMP-1 (cat. no. 54376) were from Cell Signaling Technology, Inc.) and subsequently incubated with the corresponding secondary antibodies (dilution 1:4,000) for 2 h at room temperature.

Techniques: Knockdown, Western Blot, Transfection, Control, Software, Derivative Assay, Staining, Injection, Protease Inhibitor, shRNA

Journal: International Journal of Oncology

Article Title: Diverse roles of SERPINE1 in regulating cellular proliferation and invasion

doi: 10.3892/ijo.2026.5871

Figure Lengend Snippet: SERPINE1-mediated cell cycle regulation. (A) The GO enrichment analysis of DEGs obtained from RNA-seq revealed significant changes in biological processes after SERPINE1 knockdown between shSE1 and shc groups across three cell lines. RNA-seq was performed using one biological replicate per group. (B) Heatmap of DEGs associated with the highlighted processes in (A). (C) Western blotting was performed on nuclear and cytoplasmic extracts to detect MCM3 and p-MCM3 levels (histone H3 and GAPDH were used as controls). The relative levels of p-MCM3 and MCM3 were normalized to the expression of histone H3 and GAPDH, respectively. The relative p-MCM3/MCM3 ratio was subsequently calculated. Data represent mean ± SDs of three independent experiments. (D) Flow cytometry of the cell cycle phase distribution (G 0 /G 1 , S, and G 2 /M phases). Statistical significance was determined using a two-sided Student's t test. The data are presented as the means ± SDs of three independent experiments. (E) Scatter plot of 89 cycle regulators from the protein array, highlighting SMAD3 and TP53. Blue indicates downregulated proteins (fold change ≥1.2); red indicates upregulated proteins (fold change ≤0.83); and grey indicates no change in the protein level. n=4 (F) RNA-seq data showing changes in the indicated gene expression between shSE1 and shc groups across three cell lines. RNA-seq was performed using one biological replicate per group. (G) Western blotting of the indicated proteins in C918 cells (shSE1 vs. shc). The ratios indicate the relative changes in the expression levels of the indicated proteins. Data represent mean ± SDs of three independent experiments. * P<0.05; ns, not significant. SERPINE1, serine protease inhibitor clade e member 1; GO, Gene Ontology; DEGs, differentially expressed genes; RNA-seq, RNA sequencing; shRNA, short hairpin RNA; shSE1, shRNA targeting SERPINE1; shc, shRNA scrambled control; FDR, false discovery rate; p-, phosphorylated.

Article Snippet: Following the blocking of nonspecific binding sites using 5% skimmed milk (cat. no. P0216; Beyotime Biotechnology) or bovine serum albumin (cat. no. NGP0028A; Beyotime Biotechnology) for 1 h at room temperature, the membranes were incubated with primary antibodies (incubation overnight at 4°C) against SERPINE1 (cat. no. 13801-1-AP; Proteintech Group, Inc), GAPDH (cat. no. sc-25778; Santa Cruz Biotechnology, Inc.), MCM3 (cat. no. PA5-79646; Thermo Fisher Scientific, Inc.), phosphorylated (p-)MCM3 (Ser112; cat. no. TA2362; Abmart Pharmaceutical Technology Co., Ltd.), ERK (cat. no. sc-514302; Santa Cruz Biotechnology, Inc.), uPAR (cat. no. ab10379; Abcam), Histone H3 (cat. no. 4499), p-p53 (Ser15; cat. no. 9284), p53 (cat. no. 2524), p-SMAD3 (Ser423/425; cat. no. 9520), SMAD3 (cat. no. 9523), p-Rb (Ser807/811; cat. no. 8516), Rb (cat. no. 9309), CyclinD1 (cat. no. 55506), CyclinE1 (cat. no. 20808), p21 (cat. no. 2947), p-p38 (cat. no. 4511), p38 (cat. no. 8690), p-ERK (cat. no. 4370), p-JNK (cat. no. 9255), JNK (cat. no. 9252), p-AKT (Ser473; cat. no. 4060), AKT (cat. no. 9272), HSP90α (cat. no. 4877) and MMP-1 (cat. no. 54376) were from Cell Signaling Technology, Inc.) and subsequently incubated with the corresponding secondary antibodies (dilution 1:4,000) for 2 h at room temperature.

Techniques: RNA Sequencing, Knockdown, Western Blot, Expressing, Flow Cytometry, Protein Array, Gene Expression, Protease Inhibitor, shRNA, Control

Journal: International Journal of Oncology

Article Title: Diverse roles of SERPINE1 in regulating cellular proliferation and invasion

doi: 10.3892/ijo.2026.5871

Figure Lengend Snippet: SERPINE1-mediated modulation of the ERK/p38 ratio and anoikis. (A) Upper panel: western blotting showing ERK, p-ERK, p38, and p-p38 levels. Quantitative data showed the p-ERK/ERK and p-p38/p38 ratios in SERPINE1 knockdown cells compared with control cells. Lower panel: western blotting analysis of p-AKT, AKT, p-JNK and JNK (normalized to total protein) levels in the shSE1 and shc groups. The numbers beneath the bands represent the ratios of the expression levels of the indicated proteins. Data represent mean ± SDs of three independent experiments. (B) PLAUR and HSP90AA1 mRNA and protein levels in shSE1 and shc cells analyzed using RNA-seq (left panel) and western blotting (right panel). The numbers beneath the bands represent the ratios of the expression levels of the indicated proteins and represent mean ± SDs of three independent experiments. RNA-seq was performed using one biological replicate per group. (C-D) Upper panel: (C) 231-shSE1 cells were infected with lentivirus containing PLAUR cDNA (ex-PLAUR) or the corresponding empty vector control (vec). (D) H4-shSE1 cells were transfected with siRNA targeting HSP90AA1 (si-HSP90AA1) or siNC. The expression levels of uPAR and HSP90α, as well as the activity of ERK and p38, were detected by western blotting, with GAPDH serving as the loading control. Data represent mean ± SDs of three independent experiments. Lower panel: Cell proliferation was assessed by CCK-8 assay in 231-shSE1 cells infected with ex-PLAUR or vec, and in H4-shSE1 cells transfected with si-HSP90AA1 or siNC. Statistical significance was determined using two-way ANOVA followed by Šídák's multiple comparisons test. The data are presented as the means ± SDs. n=6. (E) Morphology of shSE1 and shc cells cultured under suspension condition. (scale bar, 100 μ m). (F) Apoptotic cells cultured under suspension condition were analyzed by flow cytometry following Annexin V-PE/7AAD staining (72 h of suspension culture). Statistical significance was determined using a two-sided Student's t test. The data are presented as the means ± SDs of three independent experiments. ** P<0.01 *** P<0.001, ns, not significant. SERPINE1, serine protease inhibitor clade e member 1; p-, phosphorylated; shRNA, short hairpin RNA; shSE1, shRNA targeting SERPINE1; shc, shRNA scrambled control; PLAUR, plasminogen activator, urokinase receptor; HSP90AA1, heat shock protein 90 alpha family class a member 1; RNA-seq, RNA sequencing; vec, empty vector control; siRNA, short interfering RNA; siNC, negative control siRNA; uPAR, urokinase-type plasminogen activator receptor; HSP90α, heat shock protein 90-alpha.

Article Snippet: Following the blocking of nonspecific binding sites using 5% skimmed milk (cat. no. P0216; Beyotime Biotechnology) or bovine serum albumin (cat. no. NGP0028A; Beyotime Biotechnology) for 1 h at room temperature, the membranes were incubated with primary antibodies (incubation overnight at 4°C) against SERPINE1 (cat. no. 13801-1-AP; Proteintech Group, Inc), GAPDH (cat. no. sc-25778; Santa Cruz Biotechnology, Inc.), MCM3 (cat. no. PA5-79646; Thermo Fisher Scientific, Inc.), phosphorylated (p-)MCM3 (Ser112; cat. no. TA2362; Abmart Pharmaceutical Technology Co., Ltd.), ERK (cat. no. sc-514302; Santa Cruz Biotechnology, Inc.), uPAR (cat. no. ab10379; Abcam), Histone H3 (cat. no. 4499), p-p53 (Ser15; cat. no. 9284), p53 (cat. no. 2524), p-SMAD3 (Ser423/425; cat. no. 9520), SMAD3 (cat. no. 9523), p-Rb (Ser807/811; cat. no. 8516), Rb (cat. no. 9309), CyclinD1 (cat. no. 55506), CyclinE1 (cat. no. 20808), p21 (cat. no. 2947), p-p38 (cat. no. 4511), p38 (cat. no. 8690), p-ERK (cat. no. 4370), p-JNK (cat. no. 9255), JNK (cat. no. 9252), p-AKT (Ser473; cat. no. 4060), AKT (cat. no. 9272), HSP90α (cat. no. 4877) and MMP-1 (cat. no. 54376) were from Cell Signaling Technology, Inc.) and subsequently incubated with the corresponding secondary antibodies (dilution 1:4,000) for 2 h at room temperature.

Techniques: Western Blot, Knockdown, Control, Expressing, RNA Sequencing, Infection, Plasmid Preparation, Transfection, Activity Assay, CCK-8 Assay, Cell Culture, Suspension, Flow Cytometry, Staining, Protease Inhibitor, shRNA, Small Interfering RNA, Negative Control

Journal: International Journal of Oncology

Article Title: Diverse roles of SERPINE1 in regulating cellular proliferation and invasion

doi: 10.3892/ijo.2026.5871

Figure Lengend Snippet: Variable effects of SERPINE1 on cell migration and invasion. (A) KEGG enrichment analysis via GSEA comparing high and low SERPINE1 expression in BRCA, LGG, and SKCM (TCGA dataset). Representative images and quantification of Transwell assays of the migration (B) of shSE1 and shc cells. Representative images and quantification of Transwell invasion assays through (C) Matrigel and (D) collagen type I. Cells from three random fields in triplicate wells were counted. Scale bar, 100 μ m. Statistical significance was determined using a two-sided Student's t test. The data are presented as the means ± SDs of three independent experiments. ** P<0.01, * P<0.05. SERPINE1, serine protease inhibitor clade e member 1; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, gene set enrichment analysis; BRCA, breast cancer; LGG, low-grade glioma; SKCM, skin cutaneous melanoma; TCGA, The Cancer Genome Atlas; NES, normalized enrichment score; FDR, false discovery rate.

Article Snippet: Following the blocking of nonspecific binding sites using 5% skimmed milk (cat. no. P0216; Beyotime Biotechnology) or bovine serum albumin (cat. no. NGP0028A; Beyotime Biotechnology) for 1 h at room temperature, the membranes were incubated with primary antibodies (incubation overnight at 4°C) against SERPINE1 (cat. no. 13801-1-AP; Proteintech Group, Inc), GAPDH (cat. no. sc-25778; Santa Cruz Biotechnology, Inc.), MCM3 (cat. no. PA5-79646; Thermo Fisher Scientific, Inc.), phosphorylated (p-)MCM3 (Ser112; cat. no. TA2362; Abmart Pharmaceutical Technology Co., Ltd.), ERK (cat. no. sc-514302; Santa Cruz Biotechnology, Inc.), uPAR (cat. no. ab10379; Abcam), Histone H3 (cat. no. 4499), p-p53 (Ser15; cat. no. 9284), p53 (cat. no. 2524), p-SMAD3 (Ser423/425; cat. no. 9520), SMAD3 (cat. no. 9523), p-Rb (Ser807/811; cat. no. 8516), Rb (cat. no. 9309), CyclinD1 (cat. no. 55506), CyclinE1 (cat. no. 20808), p21 (cat. no. 2947), p-p38 (cat. no. 4511), p38 (cat. no. 8690), p-ERK (cat. no. 4370), p-JNK (cat. no. 9255), JNK (cat. no. 9252), p-AKT (Ser473; cat. no. 4060), AKT (cat. no. 9272), HSP90α (cat. no. 4877) and MMP-1 (cat. no. 54376) were from Cell Signaling Technology, Inc.) and subsequently incubated with the corresponding secondary antibodies (dilution 1:4,000) for 2 h at room temperature.

Techniques: Migration, Expressing, Protease Inhibitor

Journal: International Journal of Oncology

Article Title: Diverse roles of SERPINE1 in regulating cellular proliferation and invasion

doi: 10.3892/ijo.2026.5871

Figure Lengend Snippet: SERPINE1 regulates MMP activity. (A) MMP and TIMP levels in the supernatants of shSE1 and shc cells after 24 h of incubation and 10-fold concentration. Statistical significance was determined using a two-sided Student's t test. The data are presented as the means ± SDs. n=4. (B) Quantification of active MMP-1 and MMP-13 levels in cell lysates using fluorescence ELISA. Statistical significance was determined using two-way ANOVA followed by Šídák's multiple comparisons test. The data are presented as the means ± SDs of three independent experiments. (C) Western blotting showing the levels of the indicated proteins in H4-shSE1 cells at 72 h after transfection with the si-HSP90AA1 or siNC. The numbers beneath the bands represent the ratios of the expression levels of the indicated proteins and represent mean ± SDs of three independent experiments. (D) Representative images and quantification of Transwell invasion assays through Matrigel and collagen type I in H4-shSE1 cells transfected with the si-HSP90AA1 or siNC (scale bar, 100 μ m). Statistical significance was determined using a two-sided Student's t-test. The data are presented as the means ± SDs of three independent experiments. (E) Diagram showing the mechanisms underlying the effects of SERPINE1 on cancer proliferation and invasion. SERPINE1 knockdown influences cell proliferation and invasion through distinct signaling pathways. With respect to proliferation, SERPINE1 knockdown reduces TGF-β levels, and this reduction alters the activity of SMAD3, p53, and MCM3 to promote cell cycle progression. SERPINE1 knockdown interferes with the uPAR-mediated balance of the ERK/p38 ratio; it may also affect this ratio by modulating HSP90α expression and p38 activity, which suppress cell proliferation. In terms of invasion, SERPINE1 downregulation increases MMP-1 levels via the HSP90α-p38 pathway, thereby promoting cellular invasion. *** P<0.001, ** P<0.01, ns, not significant. SERPINE1, serine protease inhibitor clade e member 1; MMP, matrix metalloproteinases; TIMP, tissue inhibitors of metal proteases; shRNA, short hairpin RNA; shSE1, shRNA targeting SERPINE1; si, short interfering RNA; siHSP90AA1, short interfering heat shock protein 90 alpha family class a member 1; NC, negative control; uPAR, urokinase-type plasminogen activator receptor; p-, phosphorylated; MCM3, minichromosome maintenance complex component 3; HSP90α, heat shock protein 90-alpha.

Article Snippet: Following the blocking of nonspecific binding sites using 5% skimmed milk (cat. no. P0216; Beyotime Biotechnology) or bovine serum albumin (cat. no. NGP0028A; Beyotime Biotechnology) for 1 h at room temperature, the membranes were incubated with primary antibodies (incubation overnight at 4°C) against SERPINE1 (cat. no. 13801-1-AP; Proteintech Group, Inc), GAPDH (cat. no. sc-25778; Santa Cruz Biotechnology, Inc.), MCM3 (cat. no. PA5-79646; Thermo Fisher Scientific, Inc.), phosphorylated (p-)MCM3 (Ser112; cat. no. TA2362; Abmart Pharmaceutical Technology Co., Ltd.), ERK (cat. no. sc-514302; Santa Cruz Biotechnology, Inc.), uPAR (cat. no. ab10379; Abcam), Histone H3 (cat. no. 4499), p-p53 (Ser15; cat. no. 9284), p53 (cat. no. 2524), p-SMAD3 (Ser423/425; cat. no. 9520), SMAD3 (cat. no. 9523), p-Rb (Ser807/811; cat. no. 8516), Rb (cat. no. 9309), CyclinD1 (cat. no. 55506), CyclinE1 (cat. no. 20808), p21 (cat. no. 2947), p-p38 (cat. no. 4511), p38 (cat. no. 8690), p-ERK (cat. no. 4370), p-JNK (cat. no. 9255), JNK (cat. no. 9252), p-AKT (Ser473; cat. no. 4060), AKT (cat. no. 9272), HSP90α (cat. no. 4877) and MMP-1 (cat. no. 54376) were from Cell Signaling Technology, Inc.) and subsequently incubated with the corresponding secondary antibodies (dilution 1:4,000) for 2 h at room temperature.

Techniques: Activity Assay, Incubation, Concentration Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Expressing, Knockdown, Protein-Protein interactions, Protease Inhibitor, shRNA, Small Interfering RNA, Negative Control