Journal: Oncology Reports

Article Title: Regulation of the β‑catenin/LEF‑1 pathway by the siRNA knockdown of RUVBL1 expression inhibits breast cancer cell proliferation, migration and invasion

doi: 10.3892/or.2024.8855

Figure Lengend Snippet: Sequences of primers used for reverse transcription-quantitative PCR.

Article Snippet: After blocking with 5% skim milk for 2 h at room temperature and washing three times with 20% TBST, the PVDF membrane was incubated at 4°C overnight with primary antibodies RUVBL1 (IgG; 1:1,000; cat. no. ab133513; Abcam), β-catenin (IgG; 1:5,000, cat. no. 51067-2-AP; Proteintech Group, Inc.), LEF-1 (IgG; 1:1,000; cat. no. 14972-1-AP; Proteintech Group, Inc.) and cyclinD1 (IgG; 1:5,000; cat. no. 26939-1-AP; Proteintech Group, Inc.), respectively.

Techniques: Sequencing

Journal: Oncology Reports

Article Title: Regulation of the β‑catenin/LEF‑1 pathway by the siRNA knockdown of RUVBL1 expression inhibits breast cancer cell proliferation, migration and invasion

doi: 10.3892/or.2024.8855

Figure Lengend Snippet: Upregulation of RUVBL1 in BRCA tissues is associated with a poor prognosis of patients with BRCA. (A) Immunohistochemical images of RUVBL1 obtained from the Human Protein Atlas database revealed that the expression level of RUVBL1 protein was higher in BRCA tissues than in normal breast tissues. (B) RUVBL1 expression in BRCA tissues was higher than that in normal breast tissues in The Cancer Genome Atlas and GTEx databases (red, breast cancer tissues; gray, normal breast tissue). (C) Kaplan-Meier analysis was used to analyze the effects of RUVBL1 expression on the prognosis of patients with BRCA (red line, high RUVBL1 expression; black line, low expression of RUVBL1). *P<0.05. BRCA, breast cancer.

Article Snippet: After blocking with 5% skim milk for 2 h at room temperature and washing three times with 20% TBST, the PVDF membrane was incubated at 4°C overnight with primary antibodies RUVBL1 (IgG; 1:1,000; cat. no. ab133513; Abcam), β-catenin (IgG; 1:5,000, cat. no. 51067-2-AP; Proteintech Group, Inc.), LEF-1 (IgG; 1:1,000; cat. no. 14972-1-AP; Proteintech Group, Inc.) and cyclinD1 (IgG; 1:5,000; cat. no. 26939-1-AP; Proteintech Group, Inc.), respectively.

Techniques: Immunohistochemical staining, Expressing

Journal: Oncology Reports

Article Title: Regulation of the β‑catenin/LEF‑1 pathway by the siRNA knockdown of RUVBL1 expression inhibits breast cancer cell proliferation, migration and invasion

doi: 10.3892/or.2024.8855

Figure Lengend Snippet: Expression of RUVBL1 in normal mammary epithelial cells and breast cancer cells. The relative (A) mRNA, and (B and C) protein expression levels of RUVBL1 were detected in MCF-10a, MDA-MB-231 and MCF-7 cell lines using reverse transcription-quantitative PCR and western blot analysis, respectively. **P<0.01 and ****P<0.0001 vs. MCF-10a group or MCF-7 group or MDA-MB-231group.

Article Snippet: After blocking with 5% skim milk for 2 h at room temperature and washing three times with 20% TBST, the PVDF membrane was incubated at 4°C overnight with primary antibodies RUVBL1 (IgG; 1:1,000; cat. no. ab133513; Abcam), β-catenin (IgG; 1:5,000, cat. no. 51067-2-AP; Proteintech Group, Inc.), LEF-1 (IgG; 1:1,000; cat. no. 14972-1-AP; Proteintech Group, Inc.) and cyclinD1 (IgG; 1:5,000; cat. no. 26939-1-AP; Proteintech Group, Inc.), respectively.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncology Reports

Article Title: Regulation of the β‑catenin/LEF‑1 pathway by the siRNA knockdown of RUVBL1 expression inhibits breast cancer cell proliferation, migration and invasion

doi: 10.3892/or.2024.8855

Figure Lengend Snippet: Effect of si-RUVBL1 on the proliferation of breast cancer cell lines. Cell Counting Kit-8 assay was used to detect the proliferation of cells in the vehicle control group, si-NC group, and si-RUVBL1 group at different time points, and the absorbance ratio (absorbance value of detection wells/absorbance value of control wells) was calculated using the formula. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 vs. si-NC group. si-, small interfering; NC, negative control.

Article Snippet: After blocking with 5% skim milk for 2 h at room temperature and washing three times with 20% TBST, the PVDF membrane was incubated at 4°C overnight with primary antibodies RUVBL1 (IgG; 1:1,000; cat. no. ab133513; Abcam), β-catenin (IgG; 1:5,000, cat. no. 51067-2-AP; Proteintech Group, Inc.), LEF-1 (IgG; 1:1,000; cat. no. 14972-1-AP; Proteintech Group, Inc.) and cyclinD1 (IgG; 1:5,000; cat. no. 26939-1-AP; Proteintech Group, Inc.), respectively.

Techniques: Cell Counting, Control, Negative Control

Journal: Oncology Reports

Article Title: Regulation of the β‑catenin/LEF‑1 pathway by the siRNA knockdown of RUVBL1 expression inhibits breast cancer cell proliferation, migration and invasion

doi: 10.3892/or.2024.8855

Figure Lengend Snippet: Role of RUVBL1 in the migration and invasion of BRCA cells. (A and B) Scratch assay was used to detect the migration area of the cell scratch at 0, 12 and 24 h, and the results revealed that the migratory ability of the si-RUVBL1 group was decreased compared with that in the vehicle control and si-NC group. (C-F) Transwell assay was used to detect the migratory and invasive ability of the three groups of BRCA cells (vehicle control, si-NC and si-RUVBL1). (C and D) Cell staining at 0 and 48 h revealed that the migratory ability of the cells in the si-RUVBL1 group was decreased. (E and F) The results of cell staining at 0 and 48 h revealed that the invasive ability of cells in the si-RUVBL1 group was significantly reduced. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 vs. si-NC group. BRCA, breast cancer; si-, small interfering; NC, negative control; ns, not significant.

Article Snippet: After blocking with 5% skim milk for 2 h at room temperature and washing three times with 20% TBST, the PVDF membrane was incubated at 4°C overnight with primary antibodies RUVBL1 (IgG; 1:1,000; cat. no. ab133513; Abcam), β-catenin (IgG; 1:5,000, cat. no. 51067-2-AP; Proteintech Group, Inc.), LEF-1 (IgG; 1:1,000; cat. no. 14972-1-AP; Proteintech Group, Inc.) and cyclinD1 (IgG; 1:5,000; cat. no. 26939-1-AP; Proteintech Group, Inc.), respectively.

Techniques: Migration, Wound Healing Assay, Control, Transwell Assay, Staining, Negative Control

Journal: Oncology Reports

Article Title: Regulation of the β‑catenin/LEF‑1 pathway by the siRNA knockdown of RUVBL1 expression inhibits breast cancer cell proliferation, migration and invasion

doi: 10.3892/or.2024.8855

Figure Lengend Snippet: Expression of β-catenin pathway-related proteins was detected using western blot analysis. The intensity of the gray band was calculated using ImageJ software and shown in the figure. (A) The Kyoto Encyclopedia of Genes and Genomes database was used to identify the signaling pathway map of RUVBL1 (Pontin52 is the alias of the RUVBL1 gene; red box indicates the pathway-related protein). The results revealed that RUVBL1 was involved in the β-catenin/LEF-1 pathway, and its downstream genes were related to the cell cycle. (B and C) Reverse transcription-quantitative PCR and (D-F) western blot analysis were used to detect the mRNA and protein levels of β-catenin and LEF-1 in breast cancer cells of the three groups of vehicle control, si-NC and si-RUVBL1, respectively. The expression levels of β-catenin, LEF-1 and cyclinD1 in the si-RUVBL1 group were decreased in the MDA-MB-231 and MCF-7 cells. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 vs. si-NC. LEF-1, lymphoid enhancer factor-1; si-, small interfering; NC, negative control; ns, not significant.

Article Snippet: After blocking with 5% skim milk for 2 h at room temperature and washing three times with 20% TBST, the PVDF membrane was incubated at 4°C overnight with primary antibodies RUVBL1 (IgG; 1:1,000; cat. no. ab133513; Abcam), β-catenin (IgG; 1:5,000, cat. no. 51067-2-AP; Proteintech Group, Inc.), LEF-1 (IgG; 1:1,000; cat. no. 14972-1-AP; Proteintech Group, Inc.) and cyclinD1 (IgG; 1:5,000; cat. no. 26939-1-AP; Proteintech Group, Inc.), respectively.

Techniques: Expressing, Western Blot, Software, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Negative Control

Journal: Oncology Reports

Article Title: Regulation of the β‑catenin/LEF‑1 pathway by the siRNA knockdown of RUVBL1 expression inhibits breast cancer cell proliferation, migration and invasion

doi: 10.3892/or.2024.8855

Figure Lengend Snippet: RUVBL1 knockdown inhibits breast cancer cell proliferation, invasion and migration by affecting the β-catenin/LEF-1 pathway and cell cycle. LEF-1, lymphoid enhancer factor-1; siRNA, small interfering RNA.

Article Snippet: After blocking with 5% skim milk for 2 h at room temperature and washing three times with 20% TBST, the PVDF membrane was incubated at 4°C overnight with primary antibodies RUVBL1 (IgG; 1:1,000; cat. no. ab133513; Abcam), β-catenin (IgG; 1:5,000, cat. no. 51067-2-AP; Proteintech Group, Inc.), LEF-1 (IgG; 1:1,000; cat. no. 14972-1-AP; Proteintech Group, Inc.) and cyclinD1 (IgG; 1:5,000; cat. no. 26939-1-AP; Proteintech Group, Inc.), respectively.

Techniques: Knockdown, Migration, Small Interfering RNA

Journal: Breast Cancer Research : BCR

Article Title: Extracellular matrix protein 1 regulates cell proliferation and trastuzumab resistance through activation of epidermal growth factor signaling

doi: 10.1186/s13058-014-0479-6

Figure Lengend Snippet: Extracellular matrix protein 1 confers resistance toward trastuzumab. (A) Cells were seeded with Matrigel and treated with trastuzumab (Ttzm; 20 μg/ml) and recombinant human ECM1 (rhECM1; 200 ng/ml). The number of colonies 20 μm or greater in diameter was counted at 12 days (* P < 0.05, ** P < 0.005). WT, Wild type. (B) At 24 hours after cell seeding, each cell line was treated with anti-ECM1 antibody (5 μg/ml) and Ttzm (20 μg/ml) in fresh medium. After a further 48 hours, cell viability was analyzed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (* P < 0.05, ** P < 0.005, *** P < 0.0005). (C) BT-474 vector and ECM1 cells and BT-474 Ttzm-resistant (TR) control (Cont) short-hairpin (shRNA; shC) and ECM1 shRNA (shE) cells were passaged by subcutaneous injection into the lower flank of each mouse. When the tumor size increased up to 250 mm 3 , Ttzm at 20 mg/kg was administered to each mouse by intraperitoneal injection twice per week ( n = 5 or 6 for each group). (D) Circulating levels of ECM1 in serum from TR breast cancer patients were assessed by enzyme-linked immunosorbent assay (left) and Western blot analysis and compared with (right) corresponding data for Ttzm-responsive patients (* P < 0.05).

Article Snippet: The ECM1 short-hairpin RNA (shRNA) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Recombinant, Plasmid Preparation, Control, shRNA, Injection, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Extracellular matrix protein 1 regulates cell proliferation and trastuzumab resistance through activation of epidermal growth factor signaling

doi: 10.1186/s13058-014-0479-6

Figure Lengend Snippet: Relationship of extracellular matrix protein 1 level to breast cancer cell proliferation. (A) Each cell line was treated with 200 ng/ml recombinant human extracellular matrix protein 1 (rhECM1) or 5 μg/ml anti-ECM1 antibodies, and cells were counted with a hemocytometer over the course of 3 days (* P < 0.05, ** P < 0.005). Cont, Control; shC, Control short-hairpin RNA; shE/shECM1, Short-hairpin extracellular matrix protein 1 RNA; TR, Trastuzumab-resistant; Vec, Vector. (B) Cell cycles were analyzed by flow cytometry (* P < 0.05, ** P < 0.005). (C) Each cell line was passaged by subcutaneous injection into the lower flank of each mouse. Tumor volume was measured every 3 days ( n = 5 or 6 for each group). (D) Kaplan-Meier analyses for recurrence-free survival, overall survival and distant metastasis-free survival in the HER2-positive breast cancer patients. Data were obtained from the Kaplan-Meier Plotter Breast Cancer database . Overall survival data from breast cancer patients were stratified by ECM1 levels in HER2-positive patient sera ( n = 27).

Article Snippet: The ECM1 short-hairpin RNA (shRNA) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Recombinant, Control, shRNA, Plasmid Preparation, Flow Cytometry, Injection

Journal: Breast Cancer Research : BCR

Article Title: Extracellular matrix protein 1 regulates cell proliferation and trastuzumab resistance through activation of epidermal growth factor signaling

doi: 10.1186/s13058-014-0479-6

Figure Lengend Snippet: Extracellular matrix protein 1 augments epidermal growth factor signaling. (A) At 24 hours after seeding, BT-474 trastuzumab-resistant (TR) and BT-474 extracellular matrix protein 1 (ECM1)-expressing cells were treated with anti-ECM1 antibodies (ab; 5 μg/ml). Ten minutes later, cell lysates were analyzed on Western blots. (B) After serum starvation for 24 hours, cells were treated with recombinant human extracellular matrix protein 1 (rhECM1; 200 ng/ml) and epidermal growth factor (EGF; 10 ng/ml). Cell lysates were prepared at the indicated time points and analyzed on Western blots. (C) Total cell lysates were incubated with epidermal growth factor receptor (EGFR) antibodies overnight, and immunoprecipitates (IP) were analyzed on Western blots. IgG, Immunoglobulin G; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1; Vec, Vector. (D) Cells were incubated with 0.5 mg/ml EZ-Link NHS-SS-Biotin for 30 minutes at 4°C. The biotinylated proteins were precipitated by streptavidin, and the precipitates were analyzed on Western blots (IB) using ECM1 antibody (left). Cell surface labeling of ECM1 was conducted by immunostaining without permeabilization (right). Extracellular signal-regulated kinase (ERK) was used as an endogenous negative control protein. DIC, Differential Interference Contrast; GSH, Glutathione.

Article Snippet: The ECM1 short-hairpin RNA (shRNA) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Recombinant, Incubation, Control, shRNA, Plasmid Preparation, Labeling, Immunostaining, Negative Control

Journal: Breast Cancer Research : BCR

Article Title: Extracellular matrix protein 1 regulates cell proliferation and trastuzumab resistance through activation of epidermal growth factor signaling

doi: 10.1186/s13058-014-0479-6

Figure Lengend Snippet: Extracellular matrix protein 1 activates extracellular signal-regulated kinase signaling by upregulating epidermal growth factor receptor and HER3. (A) At 24 hours after cell seeding, each cell line was treated with recombinant human extracellular matrix protein 1 (rhECM1; 200 ng/ml) or anti-ECM1 antibodies (5 μg/ml) and further incubated for 48 hours. Cells lysates were then analyzed by Western blotting. Cont, Control; ERK, Extracellular signal-regulated kinase; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1 short-hairpin RNA; TR, Trastuzumab-resistant; Vec, Vector; WT, Wild type. (B) Epidermal growth factor receptor (EGFR) and HER3 mRNA levels were determined by real-time PCR using primers specific for EGFR and HER3 (** P < 0.005, *** P < 0.0005). (C) Each cell line was treated with 100 μg/ml cycloheximide (CHX). Cell lysates were prepared at the indicated time points and analyzed on Western blots. Band intensities on the blots were quantified using 1DScan software (Scanalytics, Milwaukee, WI) and plotted versus time as the ratios of EGFR/actin and HER3/actin intensities (* P < 0.05).

Article Snippet: The ECM1 short-hairpin RNA (shRNA) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Recombinant, Incubation, Western Blot, Control, shRNA, Plasmid Preparation, Real-time Polymerase Chain Reaction, Software

Journal: Breast Cancer Research : BCR

Article Title: Extracellular matrix protein 1 regulates cell proliferation and trastuzumab resistance through activation of epidermal growth factor signaling

doi: 10.1186/s13058-014-0479-6

Figure Lengend Snippet: Liquid chromatography-tandem mass spectrometry identification of proteins differentially secreted by BT-474 wild-type and trastuzumab-resistant BT-474 cells a

Article Snippet: The ECM1 short-hairpin RNA (shRNA) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Chromatography, Mass Spectrometry

Journal: Breast Cancer Research : BCR

Article Title: Extracellular matrix protein 1 regulates cell proliferation and trastuzumab resistance through activation of epidermal growth factor signaling

doi: 10.1186/s13058-014-0479-6

Figure Lengend Snippet: Extracellular matrix protein 1 stabilizes epidermal growth factor receptor and HER3 proteins through galectin-3/mucin 1. (A) Lysates from each cell line were analyzed by Western blotting. shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1 short-hairpin RNA; Vec, Vector; WT, Wild type. (B) At 24 hours after seeding, BT-474 trastuzumab-resistant (TR) cells were transfected with each small interfering RNA (siRNA: siC, Control; sigal-3, galectin-3; siMUC1, mucin 1), incubated further for 48 hours and analyzed on Western blots. (C) At 24 hours after seeding, cells were treated with recombinant human extracellular matrix protein 1 (rhECM1; 200 ng/ml) and incubated further for 48 hours. Cell lysates were then incubated with mucin 1 (MUC1), epidermal growth factor receptor (EGFR) and HER3 antibodies overnight. Immunoprecipitates (IP) were analyzed on Western blots. IgG, Immunoglobulin G. (D) Total cell lysates were incubated with MUC1, EGFR and HER3 antibodies overnight, and immunoprecipitates were then analyzed on Western blots.

Article Snippet: The ECM1 short-hairpin RNA (shRNA) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Control, shRNA, Plasmid Preparation, Transfection, Small Interfering RNA, Incubation, Recombinant

Journal: Breast Cancer Research : BCR

Article Title: Extracellular matrix protein 1 regulates cell proliferation and trastuzumab resistance through activation of epidermal growth factor signaling

doi: 10.1186/s13058-014-0479-6

Figure Lengend Snippet: Extracellular matrix protein 1 induces matrix metalloproteinase 9 transcription. (A) At 24 hours after seeding, cells were incubated with serum-free medium for a further 24 hours. Supernatant medium from each cell line was reacted with matrix metalloproteinase 9 (MMP9) substrate, and relative fluorescence units were determined at 480 to 620 nm. Cont, Control; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1 short-hairpin RNA; TR, Trastuzumab-resistant; Vec, Vector. (B) MMP9 mRNA levels were determined by real-time PCR using primers specific for MMP9 (* P < 0.05). Each cell line was transfected with an MMP9 promoter luciferase reporter construct. After 48 hours, cells were harvested, and the lysates were analyzed by dual-luciferase assay (* P < 0.05, *** P < 0.0005). (C) At 24 hours after seeding, each cell line was treated with recombinant human MMP9 (rhMMP9; 20 ng/ml), incubated further for 48 hours, and lysates from each cell were analyzed by Western blotting. EGFR, Epidermal growth factor receptor; ERK, Extracellular signal-regulated kinase; MUC1, Mucin 1; WT, Wild type. (D) Schematic model showing the role of extracellular matrix protein 1 (ECM1) in cell signaling. EGF, Epidermal growth factor.

Article Snippet: The ECM1 short-hairpin RNA (shRNA) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Incubation, Fluorescence, Control, shRNA, Plasmid Preparation, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Construct, Recombinant, Western Blot

Journal: Cell Reports Medicine

Article Title: Mechanical confinement promotes heat resistance of hepatocellular carcinoma via SP1/IL4I1/AHR axis

doi: 10.1016/j.xcrm.2023.101128

Figure Lengend Snippet: 3D thermal ablation model (A) Orthotopic HCC tumor after thermal ablation for 30 s in BALB/C nude mice. Cell viability determined by NADH staining in the non-lethal, sublethal, and lethal regions of tumor tissue after thermal ablation for 30 s. (B) A long cuboid construct (25∗10∗0.8 mm 3 ) for thermal ablation (left) monitored by infrared imaging (right). (C) Live/dead staining of MHCC-97H in the different regions of the 3D model after thermal ablation. (D) The distance from the lethal to the non-lethal region in 3D and animal models (five biological replicates). (E) Schematic diagram of a hyperthermal device (left) and its infrared imaging during operation for 15 min (right). (F and G) Live/dead staining of MHCC-97H and quantifications in the different regions under temperature gradients generated by the homemade hyperthermal device for 15 min (three biological replicates). (H) The bright field images and live/dead staining of spheroids (red arrows) in the 3D model with or without 49°C treatment for 15 min. (I) Immunofluorescence on the 3D model with or without 49°C treatment for 15 min and the animal thermal ablation model for N-cadherin (red), E-cadherin (red), F-Actin (green), and Hoechst (blue). (J) Immunohistochemistry staining of N-cadherin and E-cadherin from the animal thermal ablation model. (K) E-cadherin, N-cadherin, and MMP2 protein expression detected by western blot assay in the 3D model after different heat treatments. Mean ± standard deviation. Statistical analysis, two-tailed Student’s t test for (D), one-way ANOVA with post Tukey’s comparisons test for (G). n.s., not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Mouse anti-human MMP2 , Proteintech , Cat#66366-1-Ig; RRID: AB_2881746.

Techniques: Staining, Construct, Imaging, Generated, Immunofluorescence, Immunohistochemistry, Expressing, Western Blot, Standard Deviation, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Mechanical confinement promotes heat resistance of hepatocellular carcinoma via SP1/IL4I1/AHR axis

doi: 10.1016/j.xcrm.2023.101128

Figure Lengend Snippet:

Article Snippet: Mouse anti-human MMP2 , Proteintech , Cat#66366-1-Ig; RRID: AB_2881746.

Techniques: Virus, Recombinant, Saline, Bicinchoninic Acid Protein Assay, Imaging, Staining, Transfection, Software, Protein-Protein interactions, Microscopy, Fluorescence, Liquid Chromatography, Mass Spectrometry

Journal: Canadian journal of physiology and pharmacology

Article Title: Deciphering MMP9’s dual role in regulating SOD3 through protein–protein interactions

doi: 10.1139/cjpp-2023-0256

Figure Lengend Snippet: This figure presents the computational prediction of the interaction between matrix metalloproteinase-9 (MMP9) binding to superoxide dismutase-3 (SOD3), achieved through molecular docking using ClusPro and AutoDock software. In visualization, MMP9 is depicted in green, and SOD3 is represented in purple. The Prodigy webserver was utilized to analyze their binding affinity, revealing a strong interaction characterized by a ΔG (kcal mol−1) value of −16.2 and a Kd (M) value of 1.4e-12, indicative of high binding affinity. Further examination identified a potential cleavage site for MMP9 on SOD3 amino acid at position 173AA with a significant score of 0.906. This suggests a probable proteolytic interaction between the two proteins. The molecular interaction and the structural remodeling of these protein complexes were visualized and assessed using Pymol and ChimeraX. Collectively, the computational analysis, binding affinity evaluation, and identification of a potential cleavage site provide insights into the protein–protein interaction between MMP9 and SOD3, highlighting their intricate molecular relationship.

Article Snippet: The primary antibodies used targeted MMP9 (ProteinTech, Cat. No. 10375-2-AP, 1:1000 dilution) and SOD3 (ProteinTech, Cat. No. 14316-1-AP, 1:1000 dilution), with incubation times set as per standard procedures (overnight incubation at 4°C).

Techniques: Binding Assay, Software

Journal: Canadian journal of physiology and pharmacology

Article Title: Deciphering MMP9’s dual role in regulating SOD3 through protein–protein interactions

doi: 10.1139/cjpp-2023-0256

Figure Lengend Snippet: Matrix metalloproteinase-9 (MMP9) expression was assessed in human embryonic kidney-293 (HEK293) cells transfected with either the catalytic inactive MMP9 or the MMP9 overexpression plasmids. (A) Representative HEK293 cells post-transfection with MMP9 plasmid (green). Scale bar = 400 μm. (B) Representative Western blots of MMP9 (92 kD) protein in the transfected HEK293 cells. The experimental groups include a control set, cells transfected with the catalytically inactive (mutant) MMP9 plasmid, and cells with the MMP9 overexpressing plasmid (MMP9), which were subsequently treated with phorbol myristate acetate (PMA) to activates MMP9. Total protein levels served as the loading control for these experiments. The accompanying bar graph presents a densitometric analysis of Western blot bands, with statistical significance evaluated using one-way ANOVA followed by Tukey’s test. The significance levels are indicated as ****, P < 0.0001, and “ns” denotes non-significance. Each point represents one sample. N = 15.

Article Snippet: The primary antibodies used targeted MMP9 (ProteinTech, Cat. No. 10375-2-AP, 1:1000 dilution) and SOD3 (ProteinTech, Cat. No. 14316-1-AP, 1:1000 dilution), with incubation times set as per standard procedures (overnight incubation at 4°C).

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation, Western Blot, Control, Mutagenesis

Journal: Canadian journal of physiology and pharmacology

Article Title: Deciphering MMP9’s dual role in regulating SOD3 through protein–protein interactions

doi: 10.1139/cjpp-2023-0256

Figure Lengend Snippet: This figure presents a compelling visualization of the regulatory effects catalytic inactive matrix metalloproteinase-9 (MMP9) exerts on superoxide dismutase-3 (SOD3) through protein–protein interactions. (A) The representative Western blot image illustrates the protein levels of SOD3, approximately 30 kD, across various experimental conditions: a control group (CT), MMP9 cells activated by phorbol myristate acetate (PMA), MMP9 cells treated with a specific inhibitor, and cells transfected with a catalytically inactive MMP9 mutant plasmid. To ensure accuracy in protein quantification, total protein served as the loading control. The accompanying bar graph provides a detailed densitometric analysis of the Western blot bands, allowing for a quantitative comparison of SOD3 expression across the different treatment groups. This analysis represents data from six separate experiments (N = 6), ensuring a robust and reliable assessment of the experimental outcomes. (B) The co-immunoprecipitation results provide evidence of the interaction between MMP9 and SOD3 proteins. We conducted immunoprecipitation with an MMP9 antibody and subsequent immunoblotting with a SOD3 antibody on cell lysates. The detection of SOD3 in complexes with mutant MMP9 underscores a direct protein-protein interaction between MMP9 and SOD3. The accompanying bar graph offers a densitometric analysis of the SOD3 bands from the Western blot, quantitatively depicting the extent of this interaction. Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test, with significance levels denoted as ** for P < 0.001 and **** for P < 0.0001. ’ns’ indicates non-significant differences. This assay was repeated thrice (N = 3) to ensure data robustness. Each point represents one sample.

Article Snippet: The primary antibodies used targeted MMP9 (ProteinTech, Cat. No. 10375-2-AP, 1:1000 dilution) and SOD3 (ProteinTech, Cat. No. 14316-1-AP, 1:1000 dilution), with incubation times set as per standard procedures (overnight incubation at 4°C).

Techniques: Protein-Protein interactions, Western Blot, Control, Transfection, Mutagenesis, Plasmid Preparation, Comparison, Expressing, Immunoprecipitation

Journal: Canadian journal of physiology and pharmacology

Article Title: Deciphering MMP9’s dual role in regulating SOD3 through protein–protein interactions

doi: 10.1139/cjpp-2023-0256

Figure Lengend Snippet: The proximal ligation assay (PLA) effectively demonstrated a direct protein-protein interaction between matrix metalloproteinase-9 (MMP9) and superoxide dismutase-3 (SOD3). In this assay, we fixed the experimental cells and introduced specific antibodies targeting SOD3 and MMP9. The close proximity of these proteins was evidenced by the formation of red fluorescent dots, signifying their interaction. To confirm the assay’s specificity, we included a negative control using only one of the primary antibodies. Scale bar = 10 μm.

Article Snippet: The primary antibodies used targeted MMP9 (ProteinTech, Cat. No. 10375-2-AP, 1:1000 dilution) and SOD3 (ProteinTech, Cat. No. 14316-1-AP, 1:1000 dilution), with incubation times set as per standard procedures (overnight incubation at 4°C).

Techniques: Ligation, Negative Control

Journal: Canadian journal of physiology and pharmacology

Article Title: Deciphering MMP9’s dual role in regulating SOD3 through protein–protein interactions

doi: 10.1139/cjpp-2023-0256

Figure Lengend Snippet: The Western blots and accompanying bar graphs delineate the levels of superoxide dismutase-3 (SOD3) and matrix metalloproteinase-9 (MMP9) in the culture medium of HEK293 cells. The experiment encompassed three distinct groups: a control (CT), cells transfected with an MMP9 overexpressing plasmid followed by phorbol myristate acetate (PMA) treatment to activate MMP9, and cells transfected with a catalytically inactive mutant MMP9 plasmid. The Western blot data, presented in two sections, (A) highlight MMP9 levels and (B) focus on SOD3 levels. For each sample, total protein was utilized as the loading control. We conducted statistical analyses using one-way ANOVA followed by Tukey’s post-hoc test across the groups, with each data point representing an individual sample (N = 6). Significance levels are marked as ** (P < 0.01) and **** (P < 0.0001), with ’ns’ indicating non-significant results.

Article Snippet: The primary antibodies used targeted MMP9 (ProteinTech, Cat. No. 10375-2-AP, 1:1000 dilution) and SOD3 (ProteinTech, Cat. No. 14316-1-AP, 1:1000 dilution), with incubation times set as per standard procedures (overnight incubation at 4°C).

Techniques: Western Blot, Control, Transfection, Plasmid Preparation, Mutagenesis

Journal: Canadian journal of physiology and pharmacology

Article Title: Deciphering MMP9’s dual role in regulating SOD3 through protein–protein interactions

doi: 10.1139/cjpp-2023-0256

Figure Lengend Snippet: The schematic illustrates the distinct regulatory roles of matrix metalloproteinase-9 (MMP9) on superoxide dismutase-3 (SOD3). It showcases how active MMP9 potentially enhances SOD3 levels, likely through transcriptional upregulation. In contrast, catalytically inactive MMP9 seems to reduce SOD3 levels through direct protein-protein interactions and possibly via a unique proteolytic degradation mechanism. This differential regulation by MMP9 leads to increased secretion of SOD3 from cells in its active form, while the catalytically inactive MMP9 impedes SOD3 secretion. Phorbol myristate acetate (PMA), identified as an MMP9 activator in the schematic, plays a crucial role in modulating these interactions.

Article Snippet: The primary antibodies used targeted MMP9 (ProteinTech, Cat. No. 10375-2-AP, 1:1000 dilution) and SOD3 (ProteinTech, Cat. No. 14316-1-AP, 1:1000 dilution), with incubation times set as per standard procedures (overnight incubation at 4°C).

Techniques: Protein-Protein interactions