Journal: Investigative and Clinical Urology

Article Title: Suppression of CD81 promotes bladder cancer cell invasion through increased matrix metalloproteinase expression via extracellular signal-regulated kinase phosphorylation

doi: 10.4111/icu.2019.60.5.396

Figure Lengend Snippet: Increased expression and activity of MMP2/9 in CD81-knockdown bladder cancer cells. (A) Zymography for MMP activity. (B) Western blot analysis of MMP2 and MMP9. The culture supernatant was concentrated by an Amicon Ultra-2 Centrifugal Filter and MMP2/9 expression was analyzed using each specific antibody.

Article Snippet: Mouse polyclonal anti-CD81 antibody (Santa Cruz Biotechnology), rabbit anti-Akt antibody (Bioss Antibodies Inc., Woburn, MA, USA), rabbit anti-phospho-Akt antibody (Cell Signaling Technology, Beverly, CA, USA), mouse anti-ERK antibody (Bioss Antibodies Inc.), anti-phospho-ERK (Bioss Antibodies Inc.), rabbit anti-p38 antibody (Bioss Antibodies Inc.), rabbit anti-c-Jun N-terminal kinases (JNK) antibody (Bioss Antibodies Inc.), anti-phospho-JNK (Santa Cruz Biotechnology), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cusabio, Houston, TX, USA), rabbit anti-MMP2 antibody (Bioss Antibodies Inc.), rabbit anti-MMP9 antibody (Bioss Antibodies Inc.) were used as primary antibodies.

Techniques: Expressing, Activity Assay, Zymography, Western Blot

Journal: Investigative and Clinical Urology

Article Title: Suppression of CD81 promotes bladder cancer cell invasion through increased matrix metalloproteinase expression via extracellular signal-regulated kinase phosphorylation

doi: 10.4111/icu.2019.60.5.396

Figure Lengend Snippet: Screening for MMP2/9-associated signaling pathways in CD81-knockdown bladder cancer cells. MMP2/9-associated signaling pathways were analyzed in CD81-knockdown T24 (A) and J82 (B) bladder cancer cell lines. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping control. p-Akt, phospho-Akt; ERK, extracellular signal-regulated kinase; p-ERK, phospho-ERK; JNK, c-Jun N-terminal kinases; p-JNK, phospho-JNK.

Article Snippet: Mouse polyclonal anti-CD81 antibody (Santa Cruz Biotechnology), rabbit anti-Akt antibody (Bioss Antibodies Inc., Woburn, MA, USA), rabbit anti-phospho-Akt antibody (Cell Signaling Technology, Beverly, CA, USA), mouse anti-ERK antibody (Bioss Antibodies Inc.), anti-phospho-ERK (Bioss Antibodies Inc.), rabbit anti-p38 antibody (Bioss Antibodies Inc.), rabbit anti-c-Jun N-terminal kinases (JNK) antibody (Bioss Antibodies Inc.), anti-phospho-JNK (Santa Cruz Biotechnology), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cusabio, Houston, TX, USA), rabbit anti-MMP2 antibody (Bioss Antibodies Inc.), rabbit anti-MMP9 antibody (Bioss Antibodies Inc.) were used as primary antibodies.

Techniques:

Journal: Experimental and Therapeutic Medicine

Article Title: IL-32γ promotes integrin αvβ6 expression through the activation of NF-κB in HSCs

doi: 10.3892/etm.2017.4956

Figure Lengend Snippet: Effect of different concentrations (0, 5, 10 and 20 ng/ml) of IL-32γ on LX-2 activation phenotypes. (A) Growth curves of LX-2 demonstrated that upregulation of IL-32γ promoted proliferation of LX-2. (B) Reverse transcription-quantitative polymerase chain reaction assessing mRNA levels of α-SMA, (C) collagen I, (D) TIMP1, (E) MMP2 and (F) MMP9, representing the activation level of LX-2. (G) Western blot analysis was used to measure collagen I, MMP9, MMP2, α-SMA, TIMP1 and GAPDH expression in whole-cell extracts. Data are presented as the mean ± standard deviation of three experiments. *P<0.01 and **P<0.05 vs. 0 ng/ml IL-32γ. IL-32γ, interleukin-32γ; α-SMA, α-smooth muscle actin; TIMP1, tissue inhibitor of metalloproteinase 1; MMP, matrix metalloproteinases; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Primary antibodies were as follows: Anti-integrin αVβ6 (cat. no. ab97588; Abcam, Cambridge, MA, USA), anti-GAPDH (cat. no. KGAA002-2; Nanjing KeyGen Biotech Co., Ltd.), anti-α-SMA (cat. no. G6669; Sigma-Aldrich; Merck KGaA), collagen type I antibody (cat. no. 600-402-103; Rockland, Limerick, PA, USA), TIMP1 (cat. no. 8946), MMP2 (cat. no. 87809), MMP9 (cat. no. 13667), NF-κB: p65 (cat. no. 8242), p50 (cat. no. 3035) (all Cell Signaling Technology, Inc., Danvers, MA, USA) and IL-32γ (cat. no. 513501; Biolegend, Inc.).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Standard Deviation, Quantitative RT-PCR

Journal: BMC cancer

Article Title: Enhanced anti-cancer effect of AMTB hydrochloride via chitosan nanoparticles in pancreatic cancer.

doi: 10.1186/s12885-025-14356-w

Figure Lengend Snippet: Fig. 2 TRPM8 antagonist AMTB restricts pancreatic cancer cell migration and invasion. (A) Scratch assay showing reduced migration in AMTB-treated BxPC-3 and PANC-1 cells. (B) Transwell assay indicating decreased invasion in AMTB-treated cells. (C) Western blot analysis of EMT-related proteins and migration/invasion markers (MMP2, MMP9) in AMTB-treated cells. N = 3 (biological replicates)

Article Snippet: The relevant primary antibodies are as follows: GAPDH (5174 S, 1:1000, Cell signaling Technology [CST], Boston, USA), TRPM8 (ab85617, 1:1000, Abcam), E-cadherin (20874-1-AP, 1:20000, Proteintech, Wuhan, China), N-cadherin (AF4039, 1:500, Affinity Bioscience), Snail (CSB-PA004123, 1:1000, CUSABIO, Wuhan, China), MMP2 (CSB-PA003258, 1:1000, CUSABIO), and MMP9 (ab283575, 1:1000, Abcam).

Techniques: Migration, Wound Healing Assay, Transwell Assay, Western Blot

Journal: Advanced Science

Article Title: Aged Breast Extracellular Matrix Drives Mammary Epithelial Cells to an Invasive and Cancer‐Like Phenotype

doi: 10.1002/advs.202100128

Figure Lengend Snippet: Aged breast ECM leads to deformation of KTB21 cell spheroids and delocalization of E‐CAD from cell membrane to cytosol. A) KTB21 spheroids on the matrices monitored for 12 days. B) Microscopy images showing the spheroid presence on matrices at day 15. Top: bright field and bottom: second harmonic generation (SHG) images. Inset: image at a deeper focal plane of the dashed square. Arrowheads point to spheroids. C,D) E‐CAD, MMP2, and COL1 staining (day 15). C) E‐CAD localization in cells. Top: representative confocal microscopy images of matrices. n = 3. Bottom: plot profile of E‐CAD signal intensity across a representative cell. n = 6 cells/matrix for young and 9 for aged. D) MMP2 and COL1 staining. Top: representative confocal microscopy images of matrices. Bottom: quantification of the signal intensities. n = 3. E‐CAD and COL1: Alexa fluor 488 (green), MMP2: Alexa fluor 647 (magenta). Nucleic acid: DAPI (blue). Quantifications are performed using the Fiji software. Data are presented as the mean ± SD. Statistical test: two‐tailed student's t ‐test.

Article Snippet: Briefly, matrices at day 15 of culture were fixed with 4% PFA and permeabilized with 0.3% Triton X‐100, and then incubated for 45 min in 5% goat serum, overnight at 4 °C in mouse anti‐human E‐CAD (Abcam), mouse anti‐human COL1 (Abcam), rabbit anti‐human MMP2 (CusaBio), rabbit anti‐human MMP9 (Abcam), and mouse anti‐human LOX (LSBio) monoclonal antibodies with 1:100 dilutions, and then for 1 h in Alexa fluor 488‐labelled goat anti‐mouse IgG (Abcam), and Alexa fluor 647‐labelled goat anti‐rabbit IgG (Abcam) with 1:400 dilutions.

Techniques: Membrane, Microscopy, Staining, Confocal Microscopy, Software, Two Tailed Test

Journal: Advanced Science

Article Title: Aged Breast Extracellular Matrix Drives Mammary Epithelial Cells to an Invasive and Cancer‐Like Phenotype

doi: 10.1002/advs.202100128

Figure Lengend Snippet: LOX knockdown reverses the phenotypic and biochemical changes in KTB21 cells induced by the aged microenvironment. A) Microscopy images showing MMP2, MMP9, E‐CAD, and LOX expression, and spheroid stability on matrices (day 15). siRNAs applied between days 8–10. Fluorescence microscopy images showing MMP2 and MMP9 (magenta, Column 1), E‐CAD and LOX (green, Column 2), and nucleic acid (DAPI, blue, Column 3) staining, and merged images (Column 4). Bright field images (Column 5) showing the spheroids on matrices. n = 4. Left: young matrices, and right: aged matrices. Upper panel: scramble siRNA, and lower panel: LOX siRNA treated matrices. Arrowheads show spheroids. Results representative of two independent experiments. B) Plot profile of E‐CAD signal intensity along the diameter of representative cells in (A) showing E‐CAD localization in the cells. n = 4 images/matrix, and 8–10 cells/image. C) Cell number‐normalized signal intensities of MMP2, MMP9, E‐CAD, and LOX as quantified from (A). n = 4 matrices. Representative of three independent experiments. D,E) Heat map showing the dot blot‐based cytokine profiling of the cells on matrices (day 15). D) Cytokines and E) cancer‐associated proteins expressed by the cells. n = 3 pooled samples. Also see Tables and , Supporting Information. Quantifications were performed using the Fiji software. Data are presented as the mean ± SD. Statistical tests: one‐way ANOVA followed by Tukey's post hoc.

Article Snippet: Briefly, matrices at day 15 of culture were fixed with 4% PFA and permeabilized with 0.3% Triton X‐100, and then incubated for 45 min in 5% goat serum, overnight at 4 °C in mouse anti‐human E‐CAD (Abcam), mouse anti‐human COL1 (Abcam), rabbit anti‐human MMP2 (CusaBio), rabbit anti‐human MMP9 (Abcam), and mouse anti‐human LOX (LSBio) monoclonal antibodies with 1:100 dilutions, and then for 1 h in Alexa fluor 488‐labelled goat anti‐mouse IgG (Abcam), and Alexa fluor 647‐labelled goat anti‐rabbit IgG (Abcam) with 1:400 dilutions.

Techniques: Knockdown, Microscopy, Expressing, Fluorescence, Staining, Dot Blot, Software

Journal: Annals of Translational Medicine

Article Title: miR-145-5p targets MMP2 to protect brain injury in hypertensive intracerebral hemorrhage via inactivation of the Wnt/β-catenin signaling pathway

doi: 10.21037/atm-22-1897

Figure Lengend Snippet: Primer sequences for quantitative real-time PCR

Article Snippet: The proteins were then transferred to a polyvinylidene fluoride membrane to be probed with Bax (Immunoway, 1:1,000), Bcl-2 (Immunoway, 1:1,000), Caspase 3 (Immunoway, 1:1,000), MMP2 (Immunoway, 1:1,000), Wnt3 (Immunoway, 1:1,000), Wnt5a (Immunoway, 1:1,000), β-catenin (Proteintech, 1:1,000), TCF-4 (Proteintech, 1:1,000), LEF-1(Proteintech, 1:1,000), IL-8 (Immunoway, 1:1,000) and GAPDH (Proteintech, 1:1,000) for 24 h at 4 °C.

Techniques: Reverse Transcription

Journal: Annals of Translational Medicine

Article Title: miR-145-5p targets MMP2 to protect brain injury in hypertensive intracerebral hemorrhage via inactivation of the Wnt/β-catenin signaling pathway

doi: 10.21037/atm-22-1897

Figure Lengend Snippet: Binding of miR-145-5p to MMP2. (A) miRNAs bound to MMP2 were analyzed using miRDB, StarBase and TargetScan. Predicted binding sites between MMP2 and miR-145-5p. (B) MMP2, VEGFA and TLR4, were measured using RT-PCR. (C) Luciferase reporter assay was conducted to detect luciferase activity. (D) Expression of MMP2 was detected by western blot. Results are presented as mean ± SD. NS, no significance; *, P<0.05; **, P<0.01; ***, P<0.001. MMP2, matrix metallopeptidase 2; VEGFA, vascular endothelial growth factor-A; TLR4, Toll-like receptor 4; RT-PCR, reverse transcription-polymerase chain reaction; SD, standard deviation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control.

Article Snippet: The proteins were then transferred to a polyvinylidene fluoride membrane to be probed with Bax (Immunoway, 1:1,000), Bcl-2 (Immunoway, 1:1,000), Caspase 3 (Immunoway, 1:1,000), MMP2 (Immunoway, 1:1,000), Wnt3 (Immunoway, 1:1,000), Wnt5a (Immunoway, 1:1,000), β-catenin (Proteintech, 1:1,000), TCF-4 (Proteintech, 1:1,000), LEF-1(Proteintech, 1:1,000), IL-8 (Immunoway, 1:1,000) and GAPDH (Proteintech, 1:1,000) for 24 h at 4 °C.

Techniques: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Luciferase, Reporter Assay, Activity Assay, Expressing, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Standard Deviation, Negative Control

Journal: Annals of Translational Medicine

Article Title: miR-145-5p targets MMP2 to protect brain injury in hypertensive intracerebral hemorrhage via inactivation of the Wnt/β-catenin signaling pathway

doi: 10.21037/atm-22-1897

Figure Lengend Snippet: Effect of overexpression of miR-145-5p on the Wnt/β-catenin signaling pathway in thrombin-treated hBMECs. (A) Changes in mRNA expression of MMP2; (B) protein levels of MMP2, Wnt3, Wnt5a, β-catenin, TCF-4, LEF-1 and IL-8 in different groups detected by RT-PCR and western blot analysis, respectively. Results are presented as mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001. hBMECs, human brain microvascular endothelial cells; RT-PCR, reverse transcription-polymerase chain reaction; NC, negative control; SD, standard deviation; HICH, hypertensive intracerebral hemorrhage; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMP2, matrix metallopeptidase 2; TCF-4, transcription factor 4; LEF-1, lymphoid enhancer binding factor 1; IL-8, interleukin-8.

Article Snippet: The proteins were then transferred to a polyvinylidene fluoride membrane to be probed with Bax (Immunoway, 1:1,000), Bcl-2 (Immunoway, 1:1,000), Caspase 3 (Immunoway, 1:1,000), MMP2 (Immunoway, 1:1,000), Wnt3 (Immunoway, 1:1,000), Wnt5a (Immunoway, 1:1,000), β-catenin (Proteintech, 1:1,000), TCF-4 (Proteintech, 1:1,000), LEF-1(Proteintech, 1:1,000), IL-8 (Immunoway, 1:1,000) and GAPDH (Proteintech, 1:1,000) for 24 h at 4 °C.

Techniques: Over Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Negative Control, Standard Deviation, Binding Assay

Journal: Annals of Translational Medicine

Article Title: miR-145-5p targets MMP2 to protect brain injury in hypertensive intracerebral hemorrhage via inactivation of the Wnt/β-catenin signaling pathway

doi: 10.21037/atm-22-1897

Figure Lengend Snippet: Effect of miR-145-5p/MMP2 axis on the biological behavior of thrombin-treated hBMECs. (A) CCK8 assay of the proliferation of transfected hBMECs in each group; (B) flow cytometry to detect apoptosis of hBMECs in each group; (C) apoptosis-related proteins Bax, Bcl-2, and cleaved-caspase 3 determined by Western blot; (D) matrigel assay was used to assess the formation of capillary-like structures in hBMECs (400× magnification); (E) expression of ZO-1 and occludin was determined by immunofluorescence (400× magnification, scale bar: 50 µm); (F) vascular permeability was evaluated using FITC-labeled dextran. (G) Protein expression levels of MMP2, Wnt3, Wnt5a, β-catenin, TCF-4, LEF-1 and IL-8 in the 4 experimental groups. Results are presented as mean ± SD. **, P<0.01; ***, P<0.001. hBMECs, human brain microvascular endothelial cells; RT-PCR, reverse transcription-polymerase chain reaction; NC, negative control; SD, standard deviation; HICH, hypertensive intracerebral hemorrhage; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ZO-1, zonula occludens 1; FITC, fluorescein isothiocyanate; MMP2, matrix metallopeptidase 2; TCF-4, transcription factor 4; LEF-1, lymphoid enhancer binding factor 1; IL-8, interleukin-8.

Article Snippet: The proteins were then transferred to a polyvinylidene fluoride membrane to be probed with Bax (Immunoway, 1:1,000), Bcl-2 (Immunoway, 1:1,000), Caspase 3 (Immunoway, 1:1,000), MMP2 (Immunoway, 1:1,000), Wnt3 (Immunoway, 1:1,000), Wnt5a (Immunoway, 1:1,000), β-catenin (Proteintech, 1:1,000), TCF-4 (Proteintech, 1:1,000), LEF-1(Proteintech, 1:1,000), IL-8 (Immunoway, 1:1,000) and GAPDH (Proteintech, 1:1,000) for 24 h at 4 °C.

Techniques: CCK-8 Assay, Transfection, Flow Cytometry, Western Blot, Matrigel Assay, Expressing, Immunofluorescence, Permeability, Labeling, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Polymerase Chain Reaction, Negative Control, Standard Deviation, Binding Assay

Journal: Annals of Translational Medicine

Article Title: miR-145-5p targets MMP2 to protect brain injury in hypertensive intracerebral hemorrhage via inactivation of the Wnt/β-catenin signaling pathway

doi: 10.21037/atm-22-1897

Figure Lengend Snippet: Effect of miR-145-5p in HICH rat model. (A) The protein expression levels of ZO-1 and occludin were determined by immunofluorescence (scale bar: 50 µm); (B) the protein levels of MMP2, Wnt3, Wnt5a, β-catenin, TCF-4, LEF-1 and IL-8 in different groups detected by western blot analysis. *, P<0.05; ***, P<0.001. HICH, hypertensive intracerebral hemorrhage; DAPI, 4',6-diamidino-2-phenylindole dihydrochloride.

Article Snippet: The proteins were then transferred to a polyvinylidene fluoride membrane to be probed with Bax (Immunoway, 1:1,000), Bcl-2 (Immunoway, 1:1,000), Caspase 3 (Immunoway, 1:1,000), MMP2 (Immunoway, 1:1,000), Wnt3 (Immunoway, 1:1,000), Wnt5a (Immunoway, 1:1,000), β-catenin (Proteintech, 1:1,000), TCF-4 (Proteintech, 1:1,000), LEF-1(Proteintech, 1:1,000), IL-8 (Immunoway, 1:1,000) and GAPDH (Proteintech, 1:1,000) for 24 h at 4 °C.

Techniques: Expressing, Immunofluorescence, Western Blot