Journal: Biomedicines

Article Title: Fibroblast Activation Protein (FAP)-Mediated Cleavage of Type III Collagen Reveals Serum Biomarker Potential in Non-Small Cell Lung Cancer and Spondyloarthritis

doi: 10.3390/biomedicines12030545

Figure Lengend Snippet: Assay specificity: ( a ) Standard curves of signal inhibition relative to background (B/B0) with increasing peptide concentrations for 30aa selection peptide (std) (AGPAGAPGPAGSRGAPGPQGPRGDKGETGE), 10aa selection peptide (AGPAGAPGPA), deselection peptide (De) 1, 2, and 3 (AGPAGAAGQP, PGPAGAPGPA, and AGPQGAPGPA, respectively), elongated peptide (PAGPAGAPGPA), and truncated peptide (_GPAGAPGPA). Dotted lines indicate measurement range. ( b , c ) Biomarker measurements of C3F ( b ) and C3M ( c ) in solution of recombinant type III collagen (COL3) after incubation with/without matrix metalloproteinases 9 (MMP9) or fibroblast activation protein (FAP), with buffer as control. ( d , e ) Biomarker measurements of C3F ( d ) and C3M ( e ) in solution of recombinant type III collagen pre-cleaved with MMP13 (pre-cleaved COL3) followed by incubation with/without MMP9 or FAP, with buffer as control. Dotted lines indicate lower limit measurement range (LLMR) for C3F ( b , d ) and upper limit measurement range (ULMR) for C3M ( c , e ).

Article Snippet: In addition, we incubated recombinant type III collagen (Sigma-Aldrich, St. Louis, MO, USA, cat. #CC054) with MMP9 (Bio-Techne, Minneapolis, MN, USA, cat. #911-MP) or MMP13 (bio-techne cat. #511-MM) for 24 h and stopped the reaction with EDTA.

Techniques: Inhibition, Selection, Biomarker Assay, Recombinant, Incubation, Activation Assay

Journal: Brain

Article Title: MMP13 inhibition rescues cognitive decline in Alzheimer transgenic mice via BACE1 regulation

doi: 10.1093/brain/awy305

Figure Lengend Snippet: CL82198 inhibits BACE1 protein expression through MMP13. (A) HEK293 cells were transfected with the luciferase reporter plasmids pGL4.21-BACE1 and pGL4.10 (negative control) in the absence or presence of 5 μM CL82198 or 10 nM WAY170523 for 48 h. Luciferase assays were performed with a GloMax 96 microplate luminometer. Firefly luciferase activity was normalized to that of the plasmid pGL4.10. (B) BACE1 was knocked down with BACE1 siRNA (siBACE-1 and -6) or control siRNA (NC) in HEK293 cells for 48 h. A dramatic decrease in the amount of BACE1 protein is shown at ∼70 kD. M = protein marker. (C) HEK293 cells were treated with WAY170523 (WAY, 10 nM) for 48 h, while control cells were treated with DMSO (CTRL). (D) SH-SY5Y cells were treated with 5 μM CL82198 for 6, 12, 24 and 48 h. (E) SH-SY5Y cells were treated with 1, 2.5, 5 and 10 μM of CL82198 for 48 h. (F) SH-SY5Y cells were treated with an MMP13 neutralizing antibody (anti-MMP13, 1:500) or control rabbit IgG antibody (CTRL) for 48 h. (G–I) Mmp13 was knocked down with shRNA-1 (shMMP13-1, G) or shRNA-2 (shMMP13-2, H) in HT22 cells for 72 h or was overexpressed with an MMP13 vector in HEK293 cells for 48 h (I). CTRL = control shRNA; MOCK = control vector. (J) HEK293 cells were transfected with either the control vector (MOCK) or MMP13 vector for 48 h in the absence or presence of 5 μM CL82198 (CL). Cell lysates were subjected to western blotting analysis. Representative western blots for BACE1 are shown on the top, and quantifications are shown below. (K) Primary cultured cortical neurons were treated with 0.1, 0.5, 1, 2, 5 and 10 μM SC205756 for 48 h. (L) HEK293 cells were treated with 1 μM SC205756 for 48 h. (M) HEK-APP cells were treated with 5 µM CL82198 (CL) for 48 h. Cell lysates were prepared and subjected to western blotting analysis for APP and ADAM10. sAPPβ was analysed in conditioned media using an sAPPβ antibody. All values were normalized to CTRL or MOCK (1.0) within each experiment. The error bars are the SEM. n.s. = no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA, n = 3 or 4).

Article Snippet: MMP13 inhibitors WAY170523 (Tocris Bioscience), CL82198 (Cayman) and SC205756 (Santa Cruz Biotechnology), FGF/PDGF/VEGF RTK inhibitor 341610 (Merk Millipore), PI3 kinase inhibitors LY294002 (Sigma Aldrich), eIF4E/eIF4G interaction inhibitor 4EGI1 (Selleck), protease inhibitor MG132 (Sigma), the transcriptional inhibitor actinomycin D (ActD, Sigma) and protein biosynthesis inhibitor cycloheximide (CHX, Sigma) were dissolved in dimethyl sulphoxide (DMSO) and lysosomal inhibitor chloroquine (CQ, Sigma) was dissolved in sterilized double-distilled H 2 O.

Techniques: Expressing, Transfection, Luciferase, Negative Control, Activity Assay, Plasmid Preparation, Marker, shRNA, Western Blot, Cell Culture

Journal: Journal of Cancer

Article Title: Development and Validation of a Serum Biomarker Panel for the Detection of Esophageal Squamous Cell Carcinoma through RNA Transcriptome Sequencing

doi: 10.7150/jca.19465

Figure Lengend Snippet: Serum levels of candidate biomarkers in the preliminary screening phase. Levels of serum ADAM12, CHI3L1, MMP13 and SPP1 were compared between 40 ESCC patients (ESCC) and 40 healthy controls (HC). The Mann-Whitney U test was performed for comparisons between groups. P < 0.05 was considered statistically significant.

Article Snippet: Immunohistochemistry was performed using standard protocols with antibodies against CHI3L1 (1:50; Abcam, UK), MMP13 (1:100; R&D systems, USA), and SPP1 (1:100; Abcam, UK).

Techniques: MANN-WHITNEY

Journal: Journal of Cancer

Article Title: Development and Validation of a Serum Biomarker Panel for the Detection of Esophageal Squamous Cell Carcinoma through RNA Transcriptome Sequencing

doi: 10.7150/jca.19465

Figure Lengend Snippet: Serum ADAM12, CHI3L1, MMP13 and SPP1 in the test cohort and the validation cohort. A: Levels of biomarkers were compared between 150 ESCC patients (ESCC), 140 controls (96 healthy controls and 44 patients with benign esophageal disease) and 71 early-stage ESCC patients. Statistical analyses were performed using the Mann-Whitney U test. B: ROC curves for biomarkers and their combination (Logit(p=ESCC)=-4.583+0.017×CHI3L1+0.018×SPP1+0.821×MMP13) for the discrimination of 150 patients with ESCC and 140 controls. C: ROC curves for biomarkers and their combination for the discrimination of 71 patients with early-stage ESCC and 140 controls. D: Levels of biomarkers were compared in 169 ESCC patients (ESCC), 154 controls (80 healthy controls and 74 patients with benign esophageal disease) and 84 early-stage ESCC patients. Statistical analyses were performed using the Mann-Whitney U test. E: ROC curves for biomarkers and their combination for the discrimination of 169 patients with ESCC and 154 controls. F: ROC curves for biomarkers and their combination (Logit(p=ESCC)=-4.583+0.017×CHI3L1+0.018×SPP1+0.821×MMP13) for the discrimination of 84 patients with early-stage ESCC and 154 controls. P < 0.05 was considered statistically significant.

Article Snippet: Immunohistochemistry was performed using standard protocols with antibodies against CHI3L1 (1:50; Abcam, UK), MMP13 (1:100; R&D systems, USA), and SPP1 (1:100; Abcam, UK).

Techniques: Biomarker Discovery, MANN-WHITNEY

Journal: Journal of Cancer

Article Title: Development and Validation of a Serum Biomarker Panel for the Detection of Esophageal Squamous Cell Carcinoma through RNA Transcriptome Sequencing

doi: 10.7150/jca.19465

Figure Lengend Snippet: The diagnostic performance of ADAM12, CHI3L1, MMP13, SPP1 and their combination (Logit(p=ESCC)=-4.583+0.017×CHI3L1+0.018×SPP1+0.821×MMP13) in discriminating ESCC, early stage ESCC and controls (healthy controls and patients with esophageal benign disease) in the test cohort and validation cohort.

Article Snippet: Immunohistochemistry was performed using standard protocols with antibodies against CHI3L1 (1:50; Abcam, UK), MMP13 (1:100; R&D systems, USA), and SPP1 (1:100; Abcam, UK).

Techniques: Diagnostic Assay, Biomarker Discovery

Journal: Journal of Cancer

Article Title: Development and Validation of a Serum Biomarker Panel for the Detection of Esophageal Squamous Cell Carcinoma through RNA Transcriptome Sequencing

doi: 10.7150/jca.19465

Figure Lengend Snippet: Expression of CHI3L1, MMP13 and SPP1 mRNA or protein in ESCC cell lines and tissues and their locations in tissue. The mRNA and protein expression levels in six pairs of matched ESCC and noncancerous tissues was analyzed by real-time PCR and Western blotting, respectively (A, B), and in an immortalized esophageal epithelial cell line (NE-3) and esophageal carcinoma cell lines by real-time PCR and ELISA, respectively (C, D). The expression level was normalized to the expression of GAPDH and α-tubulin, respectively. Error bars represent standard deviations (SD) calculated from three parallel experiments. The locations of CHI3L1, MMP13 and SPP1 were determined by immunohistochemistry (E). The normal esophageal epithelial tissue showed no expression of CHI3L1, MMP13 or SPP1, respectively (E a, e, i, 200 × and b, f, j, 400×). The ESCC tissues exhibited high expression levels of these three biomarkers (E c, f, k, 200 × and d, h, l, 400×).

Article Snippet: Immunohistochemistry was performed using standard protocols with antibodies against CHI3L1 (1:50; Abcam, UK), MMP13 (1:100; R&D systems, USA), and SPP1 (1:100; Abcam, UK).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemistry