Journal: The EMBO journal

Article Title: The tumor cell-secreted matricellular protein WISP1 drives pro-metastatic collagen linearization.

doi: 10.15252/embj.2018101302

Figure Lengend Snippet: Figure 2. WISP1 is secreted by tumor cells exposed to TGFb1 and triggers Col I linearization.

Article Snippet: WISP1 ELISA WISP1 levels were detected in tissue culture-conditioned medium (24 h) with the Quantikine Mouse WISP1 ELISA Kit or with Human or Mouse WISP1 DuoSet ELISA Kit (R&D Systems).

Techniques:

Journal: The EMBO journal

Article Title: The tumor cell-secreted matricellular protein WISP1 drives pro-metastatic collagen linearization.

doi: 10.15252/embj.2018101302

Figure Lengend Snippet: Figure 5. High WISP1 gene expression levels correlate with poor prognosis in human cancers.

Article Snippet: WISP1 ELISA WISP1 levels were detected in tissue culture-conditioned medium (24 h) with the Quantikine Mouse WISP1 ELISA Kit or with Human or Mouse WISP1 DuoSet ELISA Kit (R&D Systems).

Techniques: Gene Expression

Journal: Biochimica et biophysica acta

Article Title: CCN4 induces vascular cell adhesion molecule-1 expression in human synovial fibroblasts and promotes monocyte adhesion.

doi: 10.1016/j.bbamcr.2012.12.023

Figure Lengend Snippet: Fig. 1. CCN4 increases VCAM-1 expression through αvβ5/α6β1 integrin receptor. (A) Synovial fluid was obtained from normal (n=10) or osteoarthritis patients (n=15) and examined with ELISA for the expression of CCN4. (B) Human synovial fibroblasts were cultured for 48 h, and media were collected to measure CCN4. (C–F) OASFs were incubated with various concentrations of CCN4 for 24 h or with CCN4 (30 ng/ml) for 6, 12, or 24 h. The mRNA, cell surface, and protein expression of VCAM-1 were examined by qPCR, flow cytometry, and Western blotting. (G) OASFs were pretreated for 30 min with actinomycin D or cycloheximide followed by stimulation with CCN4 for 24 h, and VCAM-1 expression was examined by qPCR. (H–J) OASFs were pretreated for 30 min with α5β1, αvβ3, αvβ5, or α6β1 integrin antibody (3 μg/ml) followed by stimulation with CCN4 for 24 h, and VCAM-1 expression was examined by qPCR, flow cytometry, and Western blotting. Results are expressed as the mean±S.E. *: pb0.05 as compared with basal level. #: pb0.05 as compared with CCN4-treated group.

Article Snippet: Synovial fluid concentrations of CCN4 were measured with an enzyme-linked immunosorbent assay (ELISA), according to the protocol provided by the manufacturer (Human CCN4 ELISA kit; R&D Systems).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Incubation, Cytometry, Western Blot

Journal: Biochimica et biophysica acta

Article Title: CCN4 induces vascular cell adhesion molecule-1 expression in human synovial fibroblasts and promotes monocyte adhesion.

doi: 10.1016/j.bbamcr.2012.12.023

Figure Lengend Snippet: Fig. 2. Syk is involved in CCN4-induced VCAM-1 expression. (A–C) OASFs were pretreated for 30 min with Syk inhibitor (10 μM) or transfected with Syk siRNA for 24 h followed by stimulation with CCN4 for 24 h, and VCAM-1 expression was examined by qPCR, flow cytometry, and Western blotting. (D–G) OASFs were incubated with CCN4 for indicated time intervals or pretreated with αvβ5 or α6β1 mAb for 30 min before incubation with CCN4 for 15 min. The Syk phosphorylation and activity were determined by Western blotting and Syk kinase assay kit. Results are expressed as the mean±S.E. *: pb0.05 as compared with basal level. #: pb0.05 as compared with CCN4-treated group.

Article Snippet: Synovial fluid concentrations of CCN4 were measured with an enzyme-linked immunosorbent assay (ELISA), according to the protocol provided by the manufacturer (Human CCN4 ELISA kit; R&D Systems).

Techniques: Expressing, Transfection, Cytometry, Western Blot, Incubation, Phospho-proteomics, Activity Assay, Kinase Assay

Journal: Biochimica et biophysica acta

Article Title: CCN4 induces vascular cell adhesion molecule-1 expression in human synovial fibroblasts and promotes monocyte adhesion.

doi: 10.1016/j.bbamcr.2012.12.023

Figure Lengend Snippet: Fig. 3. PKCδ is involved in CCN4-induced VCAM-1 expression in synovial fibroblasts. (A–C) OASFs were pretreated for 30 min with GF109203X (3 μM), rottlerin (10 μM), and Ro320432 (10 μM) or transfected with PKCδ siRNA for 24 h followed stimulated with CCN4 for 24 h, and VCAM-1 expression was examined by qPCR, flow cytometry, and Western blotting. OASFs were incubated with CCN4 for indicated time intervals (D) or pretreated with αvβ5 mAb, α6β1 mAb, or Syk inhibitor for 30 min before incubation with CCN4 for 15 min (E), and PKCδ phosphorylation was determined by Western blotting. Results are expressed as the mean±S.E. *: pb0.05 as compared with basal level. #: pb0.05 as com- pared with CCN4-treated group.

Article Snippet: Synovial fluid concentrations of CCN4 were measured with an enzyme-linked immunosorbent assay (ELISA), according to the protocol provided by the manufacturer (Human CCN4 ELISA kit; R&D Systems).

Techniques: Expressing, Transfection, Cytometry, Western Blot, Incubation, Phospho-proteomics

Journal: Biochimica et biophysica acta

Article Title: CCN4 induces vascular cell adhesion molecule-1 expression in human synovial fibroblasts and promotes monocyte adhesion.

doi: 10.1016/j.bbamcr.2012.12.023

Figure Lengend Snippet: Fig. 4. JNK is involved in CCN4-induced VCAM-1 expression in synovial fibroblasts. (A–C) OASFs were pretreated for 30 min with SP600125 (3 μM), SB203580 (10 μM), U0126 (10 μM), and PD98059 (10 μM) or transfected with JNK mutant for 24 h followed stimulated with CCN4 for 24 h, and VCAM-1 expression was examined by qPCR, flow cytometry, and Western blotting. (D) OASFs were incubated with CCN4 for indicated time intervals, and ERK, p38, and JNK phosphorylation was determined by Western blotting. Results are expressed as the mean±S.E. *: pb0.05 as compared with basal level. #: pb0.05 as compared with CCN4-treated group.

Article Snippet: Synovial fluid concentrations of CCN4 were measured with an enzyme-linked immunosorbent assay (ELISA), according to the protocol provided by the manufacturer (Human CCN4 ELISA kit; R&D Systems).

Techniques: Expressing, Transfection, Mutagenesis, Cytometry, Western Blot, Incubation, Phospho-proteomics

Journal: Biochimica et biophysica acta

Article Title: CCN4 induces vascular cell adhesion molecule-1 expression in human synovial fibroblasts and promotes monocyte adhesion.

doi: 10.1016/j.bbamcr.2012.12.023

Figure Lengend Snippet: Fig. 5. AP-1 is involved in the potentiation of VCAM-1 expression by CCN4. (A–C) OASFs were pretreated for 30 min with curcumin (3 μM) and tanshinone (5 μM) or transfected with c-Jun siRNA for 24 h then stimulated with CCN4 for 24 h, and VCAM-1 expression was examined by qPCR, flow cytometry, and Western blotting. (D) OASFs were incubated with CCN4 for indicated time intervals, and c-Jun expression in cytosol and nucleus and c-Jun phosphorylation was determined by Western blotting. Results are expressed as the mean±S.E. *: pb0.05 as compared with basal level. #: pb0.05 as compared with CCN4-treated group.

Article Snippet: Synovial fluid concentrations of CCN4 were measured with an enzyme-linked immunosorbent assay (ELISA), according to the protocol provided by the manufacturer (Human CCN4 ELISA kit; R&D Systems).

Techniques: Expressing, Transfection, Cytometry, Western Blot, Incubation, Phospho-proteomics

Journal: Biochimica et biophysica acta

Article Title: CCN4 induces vascular cell adhesion molecule-1 expression in human synovial fibroblasts and promotes monocyte adhesion.

doi: 10.1016/j.bbamcr.2012.12.023

Figure Lengend Snippet: Fig. 6. CCN4 induced AP-1 activation through integrin/Syk/PKCδ and JNK pathways. (A) OASFs were pretreated with αvβ5 mAb, α6β1 mAb, Syk inhibitor, GF109203X, and rottlerin for 30 min then stimulated with CCN4 for 120 min, the chromatin immunoprecipitation assay was then performed. (B) OASFs were pretreated with Syk inhibitor, GF109203X, rottlerin, Ro320432, and SP600125 for 30 min then stimulated with CCN4 for 30 min, and p–c-Jun expression was determined by Western blotting. (C) OASFs were pretreated with Syk inhibitor, GF109203X, rottlerin, curcumin, and tanshinone for 30 min or transfected with Syk and PKCδ siRNA or JNK mutant before exposure to CCN4. AP-1 luciferase activity was measured, and the results were normalized to the β-galactosidase activity and expressed as the mean±S.E. for three independent experiments performed in triplicate. (D&E) OASFs were incubated with various concentrations of CCN4 for 24 h or transfected with VCAM-1 siRNA for 24 h then stimulated with CCN4 for 24 h. THP-1 cells labeled with BCECF-AM were added to OASFs for 6 h, and then the THP-1 cell adherence was measured by fluorescence microscopy. Results are expressed as the mean±S.E. *: pb0.05 as com- pared with basal level. #: pb0.05 as compared with CCN4-treated group.

Article Snippet: Synovial fluid concentrations of CCN4 were measured with an enzyme-linked immunosorbent assay (ELISA), according to the protocol provided by the manufacturer (Human CCN4 ELISA kit; R&D Systems).

Techniques: Activation Assay, Chromatin Immunoprecipitation, Expressing, Western Blot, Transfection, Mutagenesis, Luciferase, Activity Assay, Incubation, Labeling, Microscopy