Journal: Translational Cancer Research

Article Title: METTL3 stabilizes SERPINE2 via the m6A modification to drive the malignant progression of gastric signet ring cell carcinoma

doi: 10.21037/tcr-24-896

Figure Lengend Snippet: High expression of SERPINE2 in human GSRCC samples and cell lines. (A,B) Volcano plots of DEGs in adenocarcinoma, partial GSRCC samples (n=2) vs . paracancerous normal gastric tissues (n=2) or adenocarcinoma tumors (n=23) (GSE33335 dataset). (C) Venn diagram of the common DEGs in GSRCC vs. normal and GSRCC vs. adenocarcinoma (GSE33335 dataset). (D) KM curves of patients with diffuse gastric cancer (n=241) stratified by SERPINE2 expression for overall survival (KM-plotter algorithm). (E) H&E staining of GSRCC samples and paracancerous normal gastric tissues. Scale bars: 100 µm. (F) Relative expression (qPCR) of SERPINE2 mRNA in GSRCC samples (n=35) and paracancerous normal gastric tissues (n=35). (G) Expression (immunoblot analysis) of SERPINE2 protein in GSRCC samples (n=5) and paracancerous normal gastric tissues (n=5). (H) Expression of SERPINE2 protein in two GSRCC cell lines (NUGC-4 and SNU-668) vs . normal GES-1 cells. GSRCC, gastric signet ring cell carcinoma; GC, gastric cancer; SERPINE2, serpin family E member 2; HR, hazard ratio; CI, confidence interval; DEGs, differentially expressed genes; KM, Kaplan-Meier; mRNA, messenger RNA; qPCR, quantitative polymerase chain reaction; IHC, immunohistochemistry.

Article Snippet: Immunoblots for SERPINE2 and METTL3 expression were conducted as described , using specific antibodies (Proteintech) recognizing SERPINE2 (rabbit polyclonal, #11303-1-AP, 1 to 800) and METTL3 (rabbit polyclonal, #15073-1-AP, 1 to 1,500).

Techniques: Expressing, Staining, Western Blot, Real-time Polymerase Chain Reaction, Immunohistochemistry

Journal: Translational Cancer Research

Article Title: METTL3 stabilizes SERPINE2 via the m6A modification to drive the malignant progression of gastric signet ring cell carcinoma

doi: 10.21037/tcr-24-896

Figure Lengend Snippet: SERPINE2 depletion alters GSRCC cell phenotypes, HUVEC tube formation and M2 macrophage polarization. (A) Expression of SERPINE2 protein in NUGC-4 and SNU-668 cells transfected with sh-SERPINE2#1, sh-SERPINE2#2 or sh-NC. (B-G) NUGC-4 and SNU-668 cells were introduced with sh-SERPINE2#1 or sh-NC. (B) In vitro cell apoptosis (flow cytometry) of transfected NUGC-4 and SNU-668 cells. (C) In vitro cell proliferation (EdU incorporation assay) of transfected NUGC-4 and SNU-668 cells. Scale bars: 100 µm. (D,E) Cell invasive and migratory capacities (Transwell assay with crystal violet staining) of transfected NUGC-4 and SNU-668 cells. Scale bars: 100 µm. (F) HUVECs were incubated with the CM from transfected NUGC-4 and SNU-668 cells and checked for tube formation ability. Scale bars: 100 µm. (G) THP-1/M0 cells were incubated with the CM from transfected NUGC-4 and SNU-668 cells and checked for the percent of the CD206 + macrophages (flow cytometry). GSRCC, gastric signet ring cell carcinoma; SERPINE2, serpin family E member 2; HUVECs, human umbilical vein endothelial cells; CM, conditioned medium; EdU, 5-ethynyl-2'-deoxyuridine; DAPI, 4',6-diamidino-2-phenylindole.

Article Snippet: Immunoblots for SERPINE2 and METTL3 expression were conducted as described , using specific antibodies (Proteintech) recognizing SERPINE2 (rabbit polyclonal, #11303-1-AP, 1 to 800) and METTL3 (rabbit polyclonal, #15073-1-AP, 1 to 1,500).

Techniques: Expressing, Transfection, In Vitro, Flow Cytometry, Transwell Assay, Staining, Incubation

Journal: Translational Cancer Research

Article Title: METTL3 stabilizes SERPINE2 via the m6A modification to drive the malignant progression of gastric signet ring cell carcinoma

doi: 10.21037/tcr-24-896

Figure Lengend Snippet: Overexpression of METTL3 in GSRCC tumors and cell lines. (A) KM curves of patients with diffuse gastric cancer (n=241) stratified by METTL3 expression for overall survival (KM-plotter algorithm). (B) Relative expression of METTL3 mRNA in GSRCC samples (n=35) and paracancerous normal gastric tissues (n=35). (C) Expression (immunoblot analysis) of METTL3 protein in GSRCC samples (n=5) and paracancerous normal gastric tissues (n=5). (D) METTL3 protein in NUGC-4 and SNU-668 GSRCC cells vs . normal GES-1 cells. (E) Expression association analysis (Pearson’s correlation test) of SERPINE2 and METTL3 in GSRCC samples (n=35). HR, hazard ratio; CI, confidence interval; GSRCC, gastric signet ring cell carcinoma; SERPINE2, serpin family E member 2; mRNA, messenger RNA; METTL3, methyltransferase-like 3; KM, Kaplan-Meier.

Article Snippet: Immunoblots for SERPINE2 and METTL3 expression were conducted as described , using specific antibodies (Proteintech) recognizing SERPINE2 (rabbit polyclonal, #11303-1-AP, 1 to 800) and METTL3 (rabbit polyclonal, #15073-1-AP, 1 to 1,500).

Techniques: Over Expression, Expressing, Western Blot

Journal: Translational Cancer Research

Article Title: METTL3 stabilizes SERPINE2 via the m6A modification to drive the malignant progression of gastric signet ring cell carcinoma

doi: 10.21037/tcr-24-896

Figure Lengend Snippet: SERPINE2 is epigenetically modulated by METTL3. (A) Prediction of a putative m6A methylation site in SERPINE2 mRNA (SRAMP tool). (B) Prediction of the binding of METTL3 and SERPINE2 mRNA (RBPsuite web). (C) Relative expression of SERPINE2 mRNA (qPCR) in NUGC-4 and SNU-668 cells transfected with sh-METTL3 or sh-NC. (D) SERPINE2 protein expression (immunoblot analysis) in NUGC-4 and SNU-668 cells transfected with sh-METTL3 or sh-NC. (E) Luciferase assays in 293T cells co-transfected with sh-METTL3 or sh-NC and WT SERPINE2 reporter construct (WT-SERPINE2) or MUT construct (MUT-SERPINE2). (F) MeRIP experiments using an anti-m6A or anti-IgG antibody with cell lysates of NUGC-4 and SNU-668 cells, and the enrichment levels of SERPINE2 mRNA (qPCR). (G) RIP experiments using an anti-METTL3 or anti-IgG antibody with cell lysates of NUGC-4 and SNU-668 cells, and quantification of the enrichment levels of SERPINE2 mRNA. (H) Relative levels of SERPINE2 mRNA (qPCR) in actinomycin D-treated NUGC-4 and SNU-668 cells after transfection of sh-METTL3 or sh-NC. GSRCC, gastric signet ring cell carcinoma; SERPINE2, serpin family E member 2; mRNA, messenger RNA; qPCR, quantitative polymerase chain reaction; METTL3, methyltransferase-like 3; m6A, N6-methyladenosine; RIP, RNA immunoprecipitation; MeRIP, methylated RIP; WT, wide-type; MUT, mutant; IgG, immunoglobulin G.

Article Snippet: Immunoblots for SERPINE2 and METTL3 expression were conducted as described , using specific antibodies (Proteintech) recognizing SERPINE2 (rabbit polyclonal, #11303-1-AP, 1 to 800) and METTL3 (rabbit polyclonal, #15073-1-AP, 1 to 1,500).

Techniques: Methylation, Binding Assay, Expressing, Transfection, Western Blot, Luciferase, Construct, Real-time Polymerase Chain Reaction, RNA Immunoprecipitation, Mutagenesis

Journal: Translational Cancer Research

Article Title: METTL3 stabilizes SERPINE2 via the m6A modification to drive the malignant progression of gastric signet ring cell carcinoma

doi: 10.21037/tcr-24-896

Figure Lengend Snippet: METTL3 depletion alters cell phenotypes via reducing SERPINE2 expression. (A-G) NUGC-4 and SNU-668 GSRCC cells were subjected to transfection of sh-NC, sh-METTL3, or sh-METTL3 along with a SERPINE2 expression plasmid. (A) SERPINE2 protein expression in transfected NUGC-4 and SNU-668 cells. (B) Cell apoptosis (flow cytometry) of transfected NUGC-4 and SNU-668 cells. (C) Cell proliferation (EdU assay) of transfected NUGC-4 and SNU-668 cells. (D,E) Cell invasive and migratory capacities (Transwell assay with crystal violet staining) of transfected NUGC-4 and SNU-668 cells. Scale bars: 100 µm. (F) HUVECs were incubated with the CM of transfected NUGC-4 and SNU-668 cells and checked for tube formation ability. (G,H) THP-1/M0 cells were incubated with the CM of transfected NUGC-4 and SNU-668 cells and checked for the percent of the CD206 + macrophages (flow cytometry) and representative images. SERPINE2, serpin family E member 2; METTL3, methyltransferase-like 3; GSRCC, gastric signet ring cell carcinoma; EdU, 5-ethynyl-2'-deoxyuridine; HUVECs, human umbilical vein endothelial cells; CM, conditioned medium.

Article Snippet: Immunoblots for SERPINE2 and METTL3 expression were conducted as described , using specific antibodies (Proteintech) recognizing SERPINE2 (rabbit polyclonal, #11303-1-AP, 1 to 800) and METTL3 (rabbit polyclonal, #15073-1-AP, 1 to 1,500).

Techniques: Expressing, Transfection, Plasmid Preparation, Flow Cytometry, EdU Assay, Transwell Assay, Staining, Incubation

Journal: Translational Cancer Research

Article Title: METTL3 stabilizes SERPINE2 via the m6A modification to drive the malignant progression of gastric signet ring cell carcinoma

doi: 10.21037/tcr-24-896

Figure Lengend Snippet: METTL3 deficiency inhibits the growth of SNU-668 xenografts in vivo . (A) Xenograft growth curves of sh-METTL3-transduced or sh-NC-infected SNU-668 cells (n=5). (B) Representative images and average weight of sh-METTL3-transduced or sh-NC-infected SNU-668 xenografts. (C) Relative expression (qPCR) of METTL3 mRNA and SERPINE2 mRNA in SNU-668 xenografts. (D) METTL3 and SERPINE2 protein expression (immunoblot analysis) in SNU-668 xenografts. (E) METTL3 and SERPINE2 expression and the Ki67-positive cell analysis (IHC assay) of SNU-668 xenografts. Scale bars: 100 µm. SERPINE2, serpin family E member 2; mRNA, messenger RNA; qPCR, quantitative polymerase chain reaction; IHC, immunohistochemistry; METTL3, methyltransferase-like 3.

Article Snippet: Immunoblots for SERPINE2 and METTL3 expression were conducted as described , using specific antibodies (Proteintech) recognizing SERPINE2 (rabbit polyclonal, #11303-1-AP, 1 to 800) and METTL3 (rabbit polyclonal, #15073-1-AP, 1 to 1,500).

Techniques: In Vivo, Infection, Expressing, Western Blot, Cell Analysis, Real-time Polymerase Chain Reaction, Immunohistochemistry

Journal: Oncotarget

Article Title: Serpin E2 promotes breast cancer metastasis by remodeling the tumor matrix and polarizing tumor associated macrophages

doi: 10.18632/oncotarget.12927

Figure Lengend Snippet: ( A ) Masson trichrome staining performed on 4T1 Ctrl and shSerpine2 tumors. Representative images show increased collagen encapsulation (blue) of SerpinE2 KD tumors. Scale bar100μm. ( B - C ) Intravital imaging using (IVI-MP) of 4T1 Ctrl and shSerpine2 tumors. (B) Representative images show collagen I fibers detected by the SHG signal (cyan) at the surface of 4T1 tumors; scale bar25μm. (C) Average SHG signal intensity was determined per 100μm z-stack; data are acquired from 19-30 separate z-stacks from 3 mice per cell line. * P < 0.00036. D. IVI-MP performed on mice bearing GFP-labeled 4T1 control and shSerpinE2 tumors. Representative images show GFP-labeled tumor cells (green), phagocytic dextran positive cells (red); SHG imaging identified collagen I fibers (cyan). Scale bars25μm. (E-F) ( E ) GFP-labeled 4T1 tumor-bearing mice were treated with control liposomes or clodronate-containing liposomes until IVI-MP was performed. Representative images are shown as in (D). ( F ) Quantification of SHG (cyan) signal intensity in 100 μm Z-stacks of tumors in treated animals. Data are mean ± SEM of measurements from 40-61 Z-stacks from at least 3 different tumors for each treatment group. * P < 0.016. All data are mean ± SEM.

Article Snippet: Anti-human SerpinE2 Ab (MAB2980) was from R&D systems.

Techniques: Staining, Encapsulation, Imaging, Labeling, Control, Liposomes

Journal: Oncotarget

Article Title: Serpin E2 promotes breast cancer metastasis by remodeling the tumor matrix and polarizing tumor associated macrophages

doi: 10.18632/oncotarget.12927

Figure Lengend Snippet: Conditioned medium (CM) from 4T1 and 168FARN ( A ) or MDA-MB435 ( B ) cultures was incubated overnight with Ab11, then bound serpinE2 was pulled-down with Protein G, and analyzed by western blot with the rodent-specific antibody, 4B3 (A), or a human-specific antibody (B). (A) The right panel shows a short exposure of serpinE2 complexes from 4T1 CM, but none in 168FARN CM (long exposure on the left), as well as an IP of the preformed tPA/serpinE2 complex. ( C ) SFM loaded with purified human serpinE2 or the preformed serpinE2/tPA complex was IP'd with Ab11 as in (A) & (B) and analyzed by western blot with a human serpinE2-specific antibody. The serpinE2 complex and uncomplexed serpinE2 are indicated with arrowheads.

Article Snippet: Anti-human SerpinE2 Ab (MAB2980) was from R&D systems.

Techniques: Incubation, Western Blot, Purification

Journal: Oncotarget

Article Title: Serpin E2 promotes breast cancer metastasis by remodeling the tumor matrix and polarizing tumor associated macrophages

doi: 10.18632/oncotarget.12927

Figure Lengend Snippet: The LRP1 receptor is expressed on tumor cells and on macrophages and we propose that serpinE2 targeting with Ab11 impacts on LRP1 signaling in both cell types. ( A ) In metastatic tumors, serpinE2-LRP1 binding stimulates ERK signaling and secretion of CCL2. Tumor cells display hyperactivation of receptor tyrosine kinases (RTKs); FGFRs, which are active in the 4T1 model, also stimulate ERK pathway activation. We speculate that in macrophages, the combination of high CCL2 levels and serpinE2-activated LRP1 promotes the M2 phenotype. Indeed, there are high levels of phagocytic, Texas-red positive M2 tumor associated macrophages (TAMs) in the metastatic tumors; they are known to be responsible for degrading the matrix during tumor development. ( B ) Ab11 or sepinE2 KD (not drawn in model) lowers ERK pathway activity and the secretion of CCL2, and stimulates TIMP1 secretion. Tyrosine kinase inhibitors (TKIs) block RTK signaling. The matrix-degrading M2 TAMs are decreased in Ab11-treated tumors. Moreover, depleting macrophages with clodronate liposomes results in deposition of a dense collagen matrix, similar to that observed in Ab11-treated tumors. Thus, we propose that the drop in CCL2, which contributes to a decrease in M2 TAMs, as well as blocking serpinE2, which skews the TAMs towards the M1 phenotype, causes the emergence of a dense collagen matrix that inhibits intravasation and metastatic dissemination.

Article Snippet: Anti-human SerpinE2 Ab (MAB2980) was from R&D systems.

Techniques: Binding Assay, Activation Assay, Activity Assay, Blocking Assay, Liposomes

Journal: Diabetes Therapy

Article Title: Elevated Serum SERPINE2 Levels are Linked to Impaired Renal Function in Patients with Type 2 Diabetes Mellitus

doi: 10.1007/s13300-025-01742-7

Figure Lengend Snippet: Serum serpin peptidase inhibitor clade E member 2 (SERPINE2) levels were compared between control subjects and patients with type 2 diabetes mellitus (T2DM). a Serum SERPINE2 levels in control subjects and patients with T2DM were detected by enzyme-linked immunosorbent assay (ELISA). The serum SERPINE2 concentration in patients with T2DM ( n = 292) was significantly higher than that in control subjects ( n = 120). Student t test was applied. b Comparison of serum SERPINE2 levels in patients with T2DM with normoalbuminuria, microalbuminuria, and macroalbuminuria. ANOVA was applied. *** P < 0.001

Article Snippet: Serum biomarker levels in control subjects and patients with T2DM were measured using ELISA kits for NGAL (QK1757, R&D Systems), KIM-1 (DSKM100, R&D Systems), TGFβ1 (DY240, R&D Systems), CTGF (DY9190-05, R&D Systems), and antibodies against SERPINE2 (AF2980, R&D Systems) [ ].

Techniques: Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Comparison

Journal: Diabetes Therapy

Article Title: Elevated Serum SERPINE2 Levels are Linked to Impaired Renal Function in Patients with Type 2 Diabetes Mellitus

doi: 10.1007/s13300-025-01742-7

Figure Lengend Snippet: Correlation between serum serpin peptidase inhibitor clade E member 2 (SERPINE2) and clinical indicators. Pearson correlation test was performed between SERPINE2 with a estimated glomerular filtration rate (eGFR), b serum creatinine (Scr), c neutrophil gelatinase-associated lipocalin (NGAL), d kidney injury molecule 1 (KIM-1), e transforming growth factor-β1 (TGFβ1), and f connective tissue growth factor (CTGF) in all patients with type 2 diabetes mellitus (T2DM)

Article Snippet: Serum biomarker levels in control subjects and patients with T2DM were measured using ELISA kits for NGAL (QK1757, R&D Systems), KIM-1 (DSKM100, R&D Systems), TGFβ1 (DY240, R&D Systems), CTGF (DY9190-05, R&D Systems), and antibodies against SERPINE2 (AF2980, R&D Systems) [ ].

Techniques: Filtration

Journal: Diabetes Therapy

Article Title: Elevated Serum SERPINE2 Levels are Linked to Impaired Renal Function in Patients with Type 2 Diabetes Mellitus

doi: 10.1007/s13300-025-01742-7

Figure Lengend Snippet: Receiver operating characteristic (ROC) curve was used to obtain the optimal cutoff value of serum serpin peptidase inhibitor clade E member 2 (SERPINE2) (278.94 pg/mL) that distinguishes the patients with type 2 diabetes mellitus (T2DM) with and without albuminuria. AUC area under the curve

Article Snippet: Serum biomarker levels in control subjects and patients with T2DM were measured using ELISA kits for NGAL (QK1757, R&D Systems), KIM-1 (DSKM100, R&D Systems), TGFβ1 (DY240, R&D Systems), CTGF (DY9190-05, R&D Systems), and antibodies against SERPINE2 (AF2980, R&D Systems) [ ].

Techniques:

Journal: Diabetes Therapy

Article Title: Elevated Serum SERPINE2 Levels are Linked to Impaired Renal Function in Patients with Type 2 Diabetes Mellitus

doi: 10.1007/s13300-025-01742-7

Figure Lengend Snippet: Schematic diagram of serpin peptidase inhibitor clade E member 2 (SERPINE2) in the development of diabetic nephropathy. Increased SERPINE2 in patients with type 2 diabetes mellitus (T2DM) with renal dysfunction promotes podocyte injury and albuminuria. SERPINE2 also enhances renal fibrosis by promoting fibrosis-related cytokines, such as transforming growth factor-β1 (TGFβ1) and connective tissue growth factor (CTGF). Therefore, the increase in SERPINE2 levels plays an aggravating role in injury of glomerular and tubular cells caused by hyperglycemia and advanced glycation end products (AGEs)

Article Snippet: Serum biomarker levels in control subjects and patients with T2DM were measured using ELISA kits for NGAL (QK1757, R&D Systems), KIM-1 (DSKM100, R&D Systems), TGFβ1 (DY240, R&D Systems), CTGF (DY9190-05, R&D Systems), and antibodies against SERPINE2 (AF2980, R&D Systems) [ ].

Techniques: