Journal: Journal of Translational Internal Medicine

Article Title: Development of a broadly potent neutralizing antibody targeting Nidogen 1 effectively inhibits cancer growth and metastasis in preclinical tumor models

doi: 10.1515/jtim-2025-0008

Figure Lengend Snippet: Upregulation of NID1 is associated with poor prognosis in HCC. A, Analysis of NID1 expression in tumor tissue (T, n = 374) and non-cancerous tissues (N, n = 49) using TCGA datasets of HCC. B, Kaplan-Meier curves comparing progression-free survival (PFS) of patients with high and low NID1 expressions in HCC stratified by median level, based on the Kaplan-Meier Plotter database ( http://kmplot.com/analysis ). C, Immunohistochemistry of NID1 was performed on TMA comprising paired T and NT tissues ( n = 80). The NID1 intensity score was analyzed. D, The pie chart illustrates the number of cases with overexpression, underexpression and no change in NID1 ( n = 80). E, Representative images of NID1 expression with intensity scores (0–3). Scale bar, 100 μm. F, Representative images of cases showing overexpression (T > NT), underexpression (T < NT) and no change (T = NT) in NID1. Scale bar, 100 μm. G, The overall IHC score of NID1 expression was plotted ( n = 80).

Article Snippet: For NID1 overexpression NID1 clone, the NID1 fragment (nucleotides 21-3123; Accession No. BC045606.1) was released from NID1/Entactin cDNA ORF Clone (Sino Biological) and subcloned into pMH-SFB cloning vector (Addgene) using gateway recombinational cloning (Invitrogen).

Techniques: Expressing, Immunohistochemistry, Over Expression

Journal: Journal of Translational Internal Medicine

Article Title: Development of a broadly potent neutralizing antibody targeting Nidogen 1 effectively inhibits cancer growth and metastasis in preclinical tumor models

doi: 10.1515/jtim-2025-0008

Figure Lengend Snippet: Generation of monoclonal anti-NID1 antibody. A, Schematic diagram illustrates the structural organization of NID1. The location of the 12-residue peptide for anti-NID1 antibody production is indicated by arrowhead. The location of epitope targets by the anti-NID1 antibody generated by the hybridoma clone is indicated by red arrowhead. B, The structural organization of NID1 and NID2. Homologous 12-residue peptide sequences in NID1 and NID2 are indicated by red arrowhead. Alignment of amino acid sequences of the selected epitope sequence between NID1 and NID2. Same residues between NID1 and NID2 are highlighted. C, Coomassie blue-stained polyacrylamide gel showing the purified anti-NID1 antibody obtained from hybridoma cells. D, Western blot analysis of NID1 expression in HLE cells stably transfected with backbone vector (XPack) and plasmid expressing NID1 (XP-NID1) using anti-NID1 antibody. E, Western blot analysis of NID1 expression in HCC cell lines using anti-NID1 antibody. F, Total cell lysates of MHCC97L cells was immunoprecipitated using anti-NID1 antibody. The immunoprecipitated proteins were subjected to immunoblotting using anti-NID1 antibody. G, Normal human hepatocyte MIHA cells were incubated with PBS or 5 μg/mL of anti-NID1 antibody. Number of cells was counted every 2 days. Data are represented as the mean ± SEM. NS, not significant from Student’s t -test.

Article Snippet: For NID1 overexpression NID1 clone, the NID1 fragment (nucleotides 21-3123; Accession No. BC045606.1) was released from NID1/Entactin cDNA ORF Clone (Sino Biological) and subcloned into pMH-SFB cloning vector (Addgene) using gateway recombinational cloning (Invitrogen).

Techniques: Residue, Generated, Sequencing, Staining, Purification, Western Blot, Expressing, Stable Transfection, Transfection, Plasmid Preparation, Immunoprecipitation, Incubation

Journal: Journal of Translational Internal Medicine

Article Title: Development of a broadly potent neutralizing antibody targeting Nidogen 1 effectively inhibits cancer growth and metastasis in preclinical tumor models

doi: 10.1515/jtim-2025-0008

Figure Lengend Snippet: Anti-NID1 antibody inhibits HCC growth and tumorigenesis. A, Immunoblots showing NID1 expression in the total cell lysate (TCL) of MHCC97 and LMHCCLM3 cells treated with anti-NID1 antibody. B, The colony forming ability of MHCC97L and MHCCLM3 cells treated with or without anti-NID1 antibody was assessed by colony formation assay. Representative images of colonies are shown (left). The number of colonies were counted and plotted (right). C, Schematic diagram of orthotopic liver implantation model. orthotopic liver implantation of luciferase-labelled MHCC97L tumor seed ( n = 7). Two weeks later, PBS, IgG and anti-NID1 antibody (400 μg/mouse) were injected intraperitoneally once a week for 21 days. Development of liver tumor was analyzed 3 weeks after liver implantation. D, At the end of experiment, tumors developed were excised and tumor size and weight were measured. Image showing the liver and tumors (left). Tumor weight (middle) and volume (right) were plotted. E, Body weight was measured regularly for 3 weeks. F, Immunohistochemistry of Ki67 staining of the excised tumors. The number of cells with positive Ki67 staining was counted. Analysis of the migration and invasiveness of MHCC97L (G) and MHCCLM3 (H) cells treated with or without anti-NID1 antibody using migration and invasion assays, respectively. Representative images of migrated and invaded cells are shown (left). Scale bar, 200 µm. Quantification of the number of migrated and invaded cells are plotted (right). I, Lung colonization of murine p53-/-; Myc hepatoblasts (1 × 105) after coinjection with anti-NID1 antibody (10 μg) via tail vein ( n = 5). Mice were subjected to bioluminescence imaging 14 days after injection. J, Bioluminescence imaging of mice at the end of the experiment. Quantification of the luciferase signal is shown. K, Bioluminescence imaging of dissected lung tissues. The intensity of luciferase signal is shown. L, Representative images of H & E staining of lung tissues. Insets show the enlarged area of the metastatic lesions. Scale bar, 150 µm. Data are represented as the mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant from Student’s t -test. P < 0.05 is considered as statistically significant.

Article Snippet: For NID1 overexpression NID1 clone, the NID1 fragment (nucleotides 21-3123; Accession No. BC045606.1) was released from NID1/Entactin cDNA ORF Clone (Sino Biological) and subcloned into pMH-SFB cloning vector (Addgene) using gateway recombinational cloning (Invitrogen).

Techniques: Western Blot, Expressing, Colony Assay, Luciferase, Injection, Immunohistochemistry, Staining, Migration, Imaging

Journal: Journal of Translational Internal Medicine

Article Title: Development of a broadly potent neutralizing antibody targeting Nidogen 1 effectively inhibits cancer growth and metastasis in preclinical tumor models

doi: 10.1515/jtim-2025-0008

Figure Lengend Snippet: Anti-NID1 antibody effectively inhibits cell growth, migration and invasion of lung cancer, breast cancer and NPC in vitro. A, Kaplan-Meier analysis of overall survival of lung cancer and breast cancer patients with high and low NID1 expression based on the TCGA dataset. B, Immunoblots showing NID1 expression in HCC827, MDA-MB-231, and C666-1 cells treated with anti-NID1 antibody. Examination of the colony forming, migratory and invasive abilities of HCC827 (C), MDA-MB-231 (D), and C666-1 (E) cells treated with or without anti-NID1 antibody. Representative images of colonies, migrated and invaded cells are shown (left). Scale bar, 200 μm. Quantification of the number of colonies, migrated and invaded cells are plotted (right). Data are represented as the mean ± SEM; ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant from Student’s t -test. P < 0.05 is considered as statistically significant.

Article Snippet: For NID1 overexpression NID1 clone, the NID1 fragment (nucleotides 21-3123; Accession No. BC045606.1) was released from NID1/Entactin cDNA ORF Clone (Sino Biological) and subcloned into pMH-SFB cloning vector (Addgene) using gateway recombinational cloning (Invitrogen).

Techniques: Migration, In Vitro, Expressing, Western Blot

Journal: Journal of Translational Internal Medicine

Article Title: Development of a broadly potent neutralizing antibody targeting Nidogen 1 effectively inhibits cancer growth and metastasis in preclinical tumor models

doi: 10.1515/jtim-2025-0008

Figure Lengend Snippet: Anti-NID1 antibody inhibits tumorigenesis of lung cancer, breast cancer and NPC in mouse models. HCC827 (A), MDA-MB-231 (D), and C666-1 (G) cells were injected subcutaneously into BALB/cAnN-nu mice. Administration of anti-NID1 antibody was started when the tumor volume reached 0.1 cm3. PBS, control IgG or anti-NID1 antibody (400 μg/mouse) were administered by intraperitoneal injection once every 3 days over a time period. B, E, F, Tumor size was measured regularly and plotted. C. F, I, At the end of experiment, tumors developed were excised and tumor size and weight were measured. Image showing the fixed excised tumors (left). Tumor weight (middle) and dimension (right) were plotted. J, Immunohistochemistry of Ki67 staining of the excised tumors. The number of Ki67 positively stained cells was analyzed. Scale bar, 100 μm. K, Body weight was measured regularly during the course of experiment. Data are represented as the mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant from Student’s t -test. P < 0.05 is considered as statistically significant.

Article Snippet: For NID1 overexpression NID1 clone, the NID1 fragment (nucleotides 21-3123; Accession No. BC045606.1) was released from NID1/Entactin cDNA ORF Clone (Sino Biological) and subcloned into pMH-SFB cloning vector (Addgene) using gateway recombinational cloning (Invitrogen).

Techniques: Injection, Control, Immunohistochemistry, Staining

Journal: Therapeutic advances in medical oncology

Article Title: Prostate cancer-associated urinary proteomes differ before and after prostatectomy.

doi: 10.1177/17588359221131532

Figure Lengend Snippet: Figure 3. Prediction of the potential tumor suppressors in the urine samples from the post-prostatectomy patients. The single and double asterisks indicate p < 0.05 and 0.01, respectively. (a) List of 41 potential tumor suppressors that were significantly enriched in the urine samples from the post-prostatectomy patients. (b) MTT-based cell viability of TRAMP prostate tumor cells, and 4T1.2 and EO771 mammary tumor cells in response to 19 recombinant proteins at 5 µg/mL. Three proteins (PRSS8, PVRL2, and NID1) were selected for further analysis. (c–e) Concentrations of prostasin (PRSS8), nectin 2 (PVRL2), and NID1 in urine from the pre and post-prostatectomy patients. NID1, nidogen 1.

Article Snippet: The overexpression of NID1 was achieved by transfecting NID1 plasmids (#12016, Addgene, Cambridge, MA, USA).

Techniques: Recombinant

Journal: Therapeutic advances in medical oncology

Article Title: Prostate cancer-associated urinary proteomes differ before and after prostatectomy.

doi: 10.1177/17588359221131532

Figure Lengend Snippet: Figure 4. Antitumor effects of PRSS8, PVRL2, and NID1 on TRAMP prostate tumor cells. The single and double asterisks indicate p < 0.05 and 0.01, respectively. (a) Elevated levels of PRSS8, PVRL2, and NID1 in the urine samples from the post-prostatectomy patients. (b and c) Downregulation of TGFβ, and upregulation of c-cas3 and p53 by the incubation of TRAMP prostate tumor cells with 1, 2 µg/mL of PRSS8 and PVRL2. (d and e) Reduction in the EdU-based proliferation and scratch-based migration of TRAMP cells by PRSS8 recombinant proteins. (f and g) Reduction in the EdU-based proliferation and scratch-based migration of TRAMP cells by PVRL2 recombinant proteins. c-cas3, cleaved caspase 3; CN, control; NID1, nidogen 1; PRSS8, prostasin; PVRL2, poliovirus receptor-related 2 or nectin 2; TGFβ, transforming growth factor beta.

Article Snippet: The overexpression of NID1 was achieved by transfecting NID1 plasmids (#12016, Addgene, Cambridge, MA, USA).

Techniques: Incubation, Migration, Recombinant, Control

Journal: Therapeutic advances in medical oncology

Article Title: Prostate cancer-associated urinary proteomes differ before and after prostatectomy.

doi: 10.1177/17588359221131532

Figure Lengend Snippet: Figure 5. Differential role of NID1 in the extracellular and intracellular domains for TRAMP prostate tumor cells. The single and double asterisks indicate p < 0.05 and 0.01, respectively. (a) Extracellular NID1 as a tumor suppressor by downregulating TGFβ and upregulating c-cas3 for apoptosis induction and p53 for tumor suppression. (b and c) Reduction in EdU-based proliferation and scratch-based motility of TRAMP cells by extracellular NID1. (d and e) Elevation in the EdU-based proliferation and scratch-based migration of TRAMP cells by the overexpression of NID1. (f) Upregulation of TGFβ and Snail, and downregulation of c-cas3 in TRAMP cells by the overexpression of NID1. (g and h) Downregulation of TGFβ and Snail, and upregulation of c-cas3 in TRAMP cells by the overexpression of PRSS8 and PVRL2. CN, control; c-cas3, cleaved caspase 3; NID1, nidogen 1; TGFβ, transforming growth factor beta.

Article Snippet: The overexpression of NID1 was achieved by transfecting NID1 plasmids (#12016, Addgene, Cambridge, MA, USA).

Techniques: Migration, Over Expression, Control

Journal: Therapeutic advances in medical oncology

Article Title: Prostate cancer-associated urinary proteomes differ before and after prostatectomy.

doi: 10.1177/17588359221131532

Figure Lengend Snippet: Figure 6. Tumor selectivity, effects on percent survival, and the putative regulatory mechanism. (a) Tumor selectivity. The tumor selectivity was defined as the ratio of ‘reduction in MTT-based viability of tumor cells’ to ‘reduction in MTT-based viability of non-tumor cells’. The double asterisk indicates p < 0.01. Two tumor cell lines (TRAMP and PC-3 cells) and three non-tumor cell lines (A5, adipose, and MC3T3 cells) were employed. Of note, N.D. means that the viability of non-tumor cells was stimulated. (b–d) Percent survival of all cancer patients with the low and high mRNA levels of PRSS8, PVRL2, and NID1. (e) Putative tumor-suppressing mechanism by urine from the post-prostatectomy patients. NID1, nidogen 1; PSA, prostate-specific antigen.

Article Snippet: The overexpression of NID1 was achieved by transfecting NID1 plasmids (#12016, Addgene, Cambridge, MA, USA).

Techniques:

Journal: Oncology Letters

Article Title: Nidogen 1 regulates proliferation and migration/invasion in murine claudin-low mammary tumor cells

doi: 10.3892/ol.2020.12313

Figure Lengend Snippet: Characteristics of murine mammary tumor cells.

Article Snippet: To reduce Nid1 expression, RJ423 cells were transfected with four unique shRNAs targeting Nid1 (cat. no. TL501474; Origene Technologies Inc.) or a control shRNA (cat. no. TR30021; Origene Technologies Inc.).

Techniques: Expressing

Journal: Oncology Letters

Article Title: Nidogen 1 regulates proliferation and migration/invasion in murine claudin-low mammary tumor cells

doi: 10.3892/ol.2020.12313

Figure Lengend Snippet: Nid1 levels in cells stably expressing Nid1 shRNA. (A) Expression levels of Nid1 mRNA relative to Hprt in parental RJ423, RJ423con, RJ423 Nid1 A and RJ423sh Nid1 D cells. The bars represent the mean value and the error bars represent the SEM. *Indicates significant difference (P<0.05) from RJ423con cells (n=6). (B) Western blot analysis of NID1 protein from cell lysates and conditioned media from RJ423, RJ423con, RJ423sh Nid1 A and RJ423sh Nid1 D cells. β-actin was used as a loading control for the cell lysates while amido black was used to ensure similar loading for the conditioned media samples. Nid1 , nidogen 1; Hprt , hypoxanthine phosphoribosyltransferase; con, control; sh, short hairpin RNA.

Article Snippet: To reduce Nid1 expression, RJ423 cells were transfected with four unique shRNAs targeting Nid1 (cat. no. TL501474; Origene Technologies Inc.) or a control shRNA (cat. no. TR30021; Origene Technologies Inc.).

Techniques: Stable Transfection, Expressing, shRNA, Western Blot

Journal: Oncology Letters

Article Title: Nidogen 1 regulates proliferation and migration/invasion in murine claudin-low mammary tumor cells

doi: 10.3892/ol.2020.12313

Figure Lengend Snippet: Impact of NID1 conditioned media on cell proliferation and apoptosis. (A) Percentage of PH3-positive cells in parental RJ423, RJ423con, RJ423sh Nid1 A and RJ423sh Nid1 D cells. (B) Percentage of apoptotic cells in RJ423, RJ423con, RJ423 Nid1 A and RJ423 Nid1 D cells. Percentage of PH3-positive cells from (C) RJ423sh Nid1 A and (D) RJ423sh Nid1 D cells treated with conditioned media from RJ423, RJ423con, RJ423sh Nid1 A or RJ423sh Nid1 D cells for 24 h. *P<0.05 vs. RJ423con cells (n=3). Nid1 , nidogen 1; con, control; sh, short hairpin RNA; PH3, phospho-histone H3.

Article Snippet: To reduce Nid1 expression, RJ423 cells were transfected with four unique shRNAs targeting Nid1 (cat. no. TL501474; Origene Technologies Inc.) or a control shRNA (cat. no. TR30021; Origene Technologies Inc.).

Techniques: shRNA

Journal: Oncology Letters

Article Title: Nidogen 1 regulates proliferation and migration/invasion in murine claudin-low mammary tumor cells

doi: 10.3892/ol.2020.12313

Figure Lengend Snippet: Impact of NID1 conditioned media on cell migration. Representative images of toluidine blue stained cells on the bottom of collagen-coated transwell inserts for (A) parental RJ423, (B) RJ423con, (C) RJ423sh Nid1 A and (D) RJ423sh Nid1 D cells (magnification, ×4). (E) Quantification of the percent area occupied by parental RJ423, RJ423con, RJ423sh Nid1 A and RJ423sh Nid1 D cells. Migration and invasion of RJ423sh Nid1 D cells when conditioned media from RJ423, RJ423con, RJ423sh Nid1 A or RJ423sh Nid1 D cells were placed (F and G) in the upper chamber of the well with the cells or (H and I) in the lower chamber. (F and H) Schematic indicating the location of the conditioned media and cells. (G and I) Quantitative data from these experiments. *P<0.05 vs. RJ423con cells (n=3). Nid1, nidogen 1; con, control; sh, short hairpin RNA; CM, conditioned media.

Article Snippet: To reduce Nid1 expression, RJ423 cells were transfected with four unique shRNAs targeting Nid1 (cat. no. TL501474; Origene Technologies Inc.) or a control shRNA (cat. no. TR30021; Origene Technologies Inc.).

Techniques: Migration, Staining, shRNA

Journal: Oncology Letters

Article Title: Nidogen 1 regulates proliferation and migration/invasion in murine claudin-low mammary tumor cells

doi: 10.3892/ol.2020.12313

Figure Lengend Snippet: Expression levels of epithelial and mesenchymal genes following Nid1 knockdown. mRNA expression levels of (A) Cdh1 , (B) Vim , (C) Snai1 , (D) Snai2 , (E) Twist1 , (F) Twist2 , (G) Zeb1 or (H) Zeb2 relative to Hprt. *P<0.05 vs. RJ423con cells (n=5). Nid1 , nidogen 1; Hprt , hypoxanthine phosphoribosyltransferase; con, control; sh, short hairpin RNA.

Article Snippet: To reduce Nid1 expression, RJ423 cells were transfected with four unique shRNAs targeting Nid1 (cat. no. TL501474; Origene Technologies Inc.) or a control shRNA (cat. no. TR30021; Origene Technologies Inc.).

Techniques: Expressing, shRNA