Journal: Journal of Neuroinflammation

Article Title: Myeloid Pannexin-1 mediates acute leukocyte infiltration and leads to worse outcomes after brain trauma

doi: 10.1186/s12974-020-01917-y

Figure Lengend Snippet: Panx1 deletion in myeloid cells reduces biomarkers of TBI. Brain tissue was isolated from ipsilateral cortex from each group and protein expression levels were determined by western blot at 6-days post-injury: a Representative western blots images showing α II spectrin and its spectrin breakdown product (SBDP 120 kDa and SBDP 140 kDa) levels at 6 days post-injury in both sham and CCI-injured Cx3cr1 -Cre:: Panx1 fl/fl and Panx1 fl/fl mice. The bottom western blot corresponds to the GAPDH, which was used as a housekeeping gene. The graph shows densitometric quantification of SBDP 120 kDa normalized to GAPDH values. Values are represented as mean ± SD ( n = 4 to 8 per group). One-way ANOVA (F 3,19 = 8.237) and Tukey’s post hoc test. b Representative western blots images showing MMP-9 levels at 6 days post-injury in both sham and CCI-injured Cx3cr1 -Cre:: Panx1 fl/fl and Panx1 fl/fl mice. The bottom western blot corresponds to the GAPDH, which was used as a housekeeping gene. The graph shows densitometric quantification of MMP-9 normalized to GAPDH values. Values are represented as mean ± SD ( n = 4 to 8 per group). Values are expressed as the mean ± SD optical density relative to GAPDH. * P < 0.05, ** P < 0.01, One-way ANOVA (F 3,17 = 8.864) and Tukey’s post hoc test.

Article Snippet: The blots were incubated in Signal Enhancer HIKARI (Nacalai Tesque INC, Japan) containing primary antibodies against alpha II spectrin (Santa Cruz Biotechnology, Dallas, TX), MMP9 (NeuroMab, San Diego, CA), IgG (Thermo Fisher Scientific, Waltham, MA) or GAPDH (Cell Signaling, Danvers, MA).

Techniques: Isolation, Expressing, Western Blot

Journal: International Journal of Oncology

Article Title: Complement regulatory protein CD46 promotes bladder cancer metastasis through activation of MMP9

doi: 10.3892/ijo.2024.5659

Figure Lengend Snippet: Expression of MMP9 by CD46 in bladder cancer cells. (A) Observation of MMP2 and MMP9 expression in five bladder cancer cell lines with CD46 overexpression, analyzed by western blotting. (B) Assessment of CD46 overexpression's impact on cell growth using an in vitro proliferation assay in cells overexpressing MMP9 due to CD46. Data are presented as mean ± SD. P-values were obtained using the U Mann Whitney test ( ns P>0.05). MMP9, matrix metalloproteinase 9; veh, vehicle; MMP2, matrix metalloproteinase 2.

Article Snippet: These sections were further incubated overnight at 4°C with primary antibodies against MMP9 (1:200; cat. no. HPA001238; Atlas Antibodies AB).

Techniques: Expressing, Over Expression, Western Blot, In Vitro, Proliferation Assay, MANN-WHITNEY

Journal: International Journal of Oncology

Article Title: Complement regulatory protein CD46 promotes bladder cancer metastasis through activation of MMP9

doi: 10.3892/ijo.2024.5659

Figure Lengend Snippet: Effects of specific inhibitors on p38 and AKT in CD46-mediated MMP9 promotion. (A) RT-qPCR of total RNA from 5637 cells to assess CD46 and MMP9 expression levels. (B) Transfection of bladder cancer cells with either AP-1-luc or MMP9-luc plasmids, followed by reporter gene transcription analysis. (C) UM-UC-3 cells underwent a reporter gene transcription assay to evaluate the effect of p38 (SB202190) and AKT (LY294002) inhibitors on CD46-mediated transcriptional activities of AP-1 (left panel) and MMP9 (right panel). (D) Treatment of both 5637 and J82 cells with indicated inhibitors for 24 h, followed by western blot analysis. Densitometric analysis of MMP9 expression is displayed as bar graphs at the bottom of each blot. Each bar represents mean ± SD. Statistical differences between groups were determined using a two-tailed unpaired Student's t-test for A and B and two-way ANOVA with Bonferroni multiple comparisons test for C and D. * P<0.01, ** P<0.001, *** P<0.0001 vs. control cells. veh, vehicle; AKT, protein kinase B; MMP9, matrix metalloproteinase 9; RT-qPCR, reverse transcription-quantitative PCR; AP-1, activator protein 1.

Article Snippet: These sections were further incubated overnight at 4°C with primary antibodies against MMP9 (1:200; cat. no. HPA001238; Atlas Antibodies AB).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Transcription Assay, Western Blot, Two Tailed Test, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Oncology

Article Title: Complement regulatory protein CD46 promotes bladder cancer metastasis through activation of MMP9

doi: 10.3892/ijo.2024.5659

Figure Lengend Snippet: Effects of CD46 on the regulation of migratory and invasive potential in bladder cancer cells. (A) Media from specified cells were collected for analysis. Non-reducing gelatin SDS-PAGE was used to demonstrate gelatinase activity (top panel) and western blot analysis was conducted to show secreted CD46 (lower panel). (B) Media from cells were analyzed by ELISA assay to show secreted MMP9. (C) Bright-field images showing scratched areas (marked by white lines) in confluent monolayers of cancer cells overexpressing CD46 at different time points (left panel). Relative wound closure was assessed by measuring the width of the wounds compared with control cells (right panel). (D) Transwell assays to measure cell migration and invasion in 5637 and J82 cells transfected with CD46 (left panel). Quantification of cell migration and invasion was conducted (right panel). Each bar represents mean ± SD. All scale bars, 200 µ m. Differences between groups were assessed using a two-tailed unpaired Student's t-test. * P<0.01, ** P<0.001, *** P<0.0001 vs. control cells. veh, vehicle; MMP9, matrix metalloproteinase 9; MMP2, matrix metalloproteinase 2.

Article Snippet: These sections were further incubated overnight at 4°C with primary antibodies against MMP9 (1:200; cat. no. HPA001238; Atlas Antibodies AB).

Techniques: SDS Page, Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Migration, Transfection, Two Tailed Test

Journal: International Journal of Oncology

Article Title: Complement regulatory protein CD46 promotes bladder cancer metastasis through activation of MMP9

doi: 10.3892/ijo.2024.5659

Figure Lengend Snippet: Effects of CD46 on experimental lung metastasis in a murine bladder cancer model. Nude mice were injected intravenously with 5637 cells. After 6 weeks, they were sacrificed for analysis of metastatic lung tumor nodules. (A) Optical images of lung metastasis in athymic nude mice injected with 5637-veh and 5637-CD46 cells (n=7 each group). (B) Quantification of lung tumor nodules in each mouse group. Each dot represents an individual mouse. (C) Histological examination of lungs from CD46-5637 and veh-5637 injected mice (a-c). High magnification images of corresponding lungs (d-f) and (g and h) immunohistochemical images for MMP9 expression in a and b. Scale bar, 2 mm (a-c), 200 µ m (d-h). (D) ELISA analysis of sera from mice before termination for serum MMP9 levels. Data represented as mean ± SD. Each dot represents an individual mouse. Statistical differences determined by paired two-tailed Student's t-test. veh, vehicle; MMP9, matrix metalloproteinase 9.

Article Snippet: These sections were further incubated overnight at 4°C with primary antibodies against MMP9 (1:200; cat. no. HPA001238; Atlas Antibodies AB).

Techniques: Injection, Immunohistochemical staining, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: International Journal of Oncology

Article Title: Complement regulatory protein CD46 promotes bladder cancer metastasis through activation of MMP9

doi: 10.3892/ijo.2024.5659

Figure Lengend Snippet: Model of CD46-induced MMP9 upregulation in bladder cancer cells. This figure proposes a model for CD46-mediated promotion of bladder cancer metastasis. Transmembrane receptor CD46 activates hyperphosphorylation of p38 MAPK and PI3K/AKT, leading to c-Jun activation in the AP-1 complex at the MMP9 promoter. Increased MMP9 secretion into the extracellular matrix enhances migration and invasion of bladder cancer cells. Secreted MMP9 may also cleave the extracellular domain of CD46, potentially regulating overstimulation of CD46-mediated MMP9 expression. The model was created using BioRender.com . MMP9, matrix metalloproteinase 9; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; AP-1, activator protein 1.

Article Snippet: These sections were further incubated overnight at 4°C with primary antibodies against MMP9 (1:200; cat. no. HPA001238; Atlas Antibodies AB).

Techniques: Activation Assay, Migration, Expressing