Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cartilage intermediate layer protein inhibits ligamentum flavum hypertrophy mediated by TGF-β1/SMAD3/SERPINE2 signaling pathway

doi: 10.1007/s00018-025-06051-7

Figure Lengend Snippet: CILP attenuates LF fibrosis via the TGF-β1/SMAD3/SERPINE2 axis. ( A ) A violin plot depicted differential SERPINE2 expression between Non-LFH and LFH groups (n = 6 per group), showing significant differences. ( B ) IHC of SERPINE2 in LF samples from Non-LFH and LFH groups (n = 6 per group). Scale bar: 50 µm. ( C , D ) Western blotting revealed elevated SERPINE2 protein levels between Non-LFH and LFH groups (n = 6 per group), with relative protein expression quantified. And mRNA concentration showed consistent results. ( E , F ) Western blotting demonstrated SERPINE2 expression in LF cells treated with TGF-β1 at 0, 8, 16, and 24 h time points, with relative protein expression quantified. And mRNA concentration showed consistent results. ( G ) Western blotting assessed protein expression levels of CILP, Collagen I, p-SMAD3, and α-SMA in LF cells subjected to various SERPINE2 treatments. ( H ) qRT-PCR assessed transcriptional profiles of CILP, ACTA2 (α-SMA), and COL1A2 across experimental conditions in LF cells. ( I ) WB analysis evaluated p-SMAD3 and fibrotic protein levels 48 hours post-transfection with CILP-overexpressing plasmids under different conditions in LF cells. ( J ) WB analysis examined p-SMAD3 and fibrotic protein expression 48 hours post-transfection with CILP-silencing plasmids under various regimens. Significance levels are denoted as follows: ns, not significant;* P < 0.05;** P < 0.01;*** P < 0.01

Article Snippet: In further experiments, cells were exposed to varying concentrations of CILP protein (10, 25, 50 ng/ml, RPC382Hu01, Cloud-Clone, TX, USA) and/or SERPINE2 protein (25 μg/ml, HY- P71085 , MCE) for 24 h. The plasmids and siRNA were obtained from Hanbio Biotechnology Co., Ltd, located in Shanghai, China.

Techniques: Expressing, Western Blot, Concentration Assay, Quantitative RT-PCR, Transfection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cartilage intermediate layer protein inhibits ligamentum flavum hypertrophy mediated by TGF-β1/SMAD3/SERPINE2 signaling pathway

doi: 10.1007/s00018-025-06051-7

Figure Lengend Snippet: CILP attenuates TGF-β1-mediated LFH in vivo. ( A - D ) IHC analysis of SERPINE2, TGF-β1, Collagen I, and p-SMAD3 in mouse tissues, with quantification of positive cell percentages. Scale bar: 50 µm. Significance levels are denoted as follows: ns, not significant;* P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: In further experiments, cells were exposed to varying concentrations of CILP protein (10, 25, 50 ng/ml, RPC382Hu01, Cloud-Clone, TX, USA) and/or SERPINE2 protein (25 μg/ml, HY- P71085 , MCE) for 24 h. The plasmids and siRNA were obtained from Hanbio Biotechnology Co., Ltd, located in Shanghai, China.

Techniques: In Vivo

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