Journal: International Journal of Biomedical Science : IJBS

Article Title: ESM-1 siRNA Knockdown Decreased Migration and Expression of CXCL3 in Prostate Cancer Cells

doi:

Figure Lengend Snippet: ESM-1 and CXC-chemokine expression in PC-3 cells relative to non-tumorigenic PWR-1E cells. A, Transcript expression levels for ESM-1, ELR+ CXC chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8), and ELR− CXC chemokines (CXCL4, CXCL9, CXCL10, CXCL11, CXCL12, CXCL14), were analyzed in the cell lines by RT-qPCR. Relative changes in gene expression for each target gene were calculated using the comparative CT method (2 –ΔΔCT ) normalizing to the expression levels of reference genes (HPRT-1 and β -actin). Greater than twofold expression changes were regarded as significant and are marked by asterisks; B, Protein levels were measured using quantitative commercial enzyme-linked immunosorbent assay/ELISA for ESM-1 and CXC chemokines, according to the manufacturer's instructions. Chemokines evaluated were CXCL1/GROα, CXCL3, CXCL8/IL8, CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC. ESM-1 and CXC chemokine levels in cell supernatants are shown as mean ± SEM.

Article Snippet: CXCL1/GROα, CXCL8/IL8, CXCL9/MIG, CXCL10/ IP-10, and CXCL11/I-TAC protein levels were determined using the Human Common Chemokines Multi-Analyte ELISArray Kit (SABiosciences, Qiagen, Valencia, CA, USA), while CXCL3 protein level was determined with a single ELISA assay kit (Cat #: MBS910472, Mybiosource, San Diego, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: Mucosal immunology

Article Title: The Matricellular Protein CCN1 Promotes Mucosal Healing in Murine Colitis through IL-6

doi: 10.1038/mi.2015.19

Figure Lengend Snippet: (A) IL-6 mRNA in the colon of wild type and Ccn1 dm/dm mice treated with 3.5% DSS for 5 days was measured by qRT-PCR, and normalized to healthy wild type colon with cyclophilin E as internal reference. Data shown as mean ± SD; n =3–4; n.s., not significant. ** P <0.003. (B) IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-12, IL-17α, IFN- γ , TNFα, and GM-CSF protein was measured by ELISA in the serum of wild type mice at indicated times after a single injection i.p. of purified WT-CCN1 protein (5 or 20 μg), or (C) 24 hours after i.p. injection of 10 μg of WT-CCN1, DM-CCN1 protein, or PBS ( n =4). (D) IL-6 was measured from the serum of 3.5% DSS-challenged wild type and Ccn1 dm/dm mice with and without CCN1 treatment by ELISA. Data are presented as mean ± SEM; n =7; * P <0.05.

Article Snippet: ELISA was conducted using IL-6 Quantikine ELISA kit (R&D); serum protein levels were quantified using Inflammatory Cytokine and Chemokine Multi-Analyte ELISArray and IL-6 single analyte ELISA kits (Qiagen) with manufacturer’s protocol.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Injection, Purification

Journal: Mucosal immunology

Article Title: The Matricellular Protein CCN1 Promotes Mucosal Healing in Murine Colitis through IL-6

doi: 10.1038/mi.2015.19

Figure Lengend Snippet: (A) IL-6 mRNA was measured by qRT-PCR and (B) IL-6 protein from conditioned media was measured by ELISA in serum-starved (overnight) I13.35 macrophages treated with 5 μg/ml of purified WT-CCN1 or DM-CCN1, or BSA for 24 hrs. (C) I13.35 macrophages were incubated with blocking mAbs against integrin α M (50 μg/ml) or β 2 (50 μg/ml) 1 hr prior to treatment with WT-CCN1 for 24 hrs. IL-6 mRNA was measured by qRT-PCR. (D) IL-6 mRNA was measured by qRT-PCR and (E) IL-6 protein from conditioned media was measured by ELISA in serum-starved 18Co fibroblasts treated with 5 μg/ml of purified WT-CCN1, DM-CCN1, BSA, or 25 ng/ml of TNFα for 24 hrs. (F) 18Co fibroblasts were incubated with blocking mAb against integrin α 6 for 1 hr, then treated with WT-CCN1, TNFα, or BSA for 24 hrs as above. IL-6 mRNA was measured by qRT-PCR. Data shown as mean ± SD of triplicate experiments.

Article Snippet: ELISA was conducted using IL-6 Quantikine ELISA kit (R&D); serum protein levels were quantified using Inflammatory Cytokine and Chemokine Multi-Analyte ELISArray and IL-6 single analyte ELISA kits (Qiagen) with manufacturer’s protocol.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Purification, Incubation, Blocking Assay

Journal: Mucosal immunology

Article Title: The Matricellular Protein CCN1 Promotes Mucosal Healing in Murine Colitis through IL-6

doi: 10.1038/mi.2015.19

Figure Lengend Snippet: (A) Ccn1 dm/dm mice ( n =20) were injected i.p. with rIL-6 (100 ng) or vehicle control daily for 5 consecutive days after each cycle of 5% DSS feeding and were monitored for survival. ** P <0.01 (log-rank test). (B) Body weight change was monitored daily in 3.5% DSS-challenged Ccn1 dm/dm mice treated with 100 ng of rIL-6 or vehicle control for 5 consecutive days after DSS feeding. (C) Mucosal damage in mice treated in (B) was evaluated by H&E staining of paraffin-embedded sections of the distal colon. Histological scores are shown on the right ( n =3–4; * P <0.05). Scale bar=100 μm. (D) Wild type and Ccn1 dm/dm mice were challenged with 3.5% DSS, and Ccn1 dm/dm mice were further treated with WT-CCN1 (5 μg) or rIL-6 (100 ng) as indicated. PCNA and E-cadherin was detected by immunofluorescence microscopy to assess IEC proliferation. DAPI was used for counterstaining. Quantitation of PCNA staining is shown on right ( n =3–4; * P <0.05, ** P <0.01). Scale bar=50 μm. (E) YAMC cells were treated with BSA, IL-6 (50 or 100 ng/ml), or EGF (100 ng/ml) for 24 hrs and stained for Ki67 by immunofluorescence and quantitated. Where indicated, YAMC cells were pre-incubated with blocking polyclonal antibodies for IL-6 receptor α (IL-6Rα) or IgG for 1 hr before IL-6 addition. Data are shown as mean ± SD; * P <0.05, ** P <0.01. Scale bar=300 μm.

Article Snippet: ELISA was conducted using IL-6 Quantikine ELISA kit (R&D); serum protein levels were quantified using Inflammatory Cytokine and Chemokine Multi-Analyte ELISArray and IL-6 single analyte ELISA kits (Qiagen) with manufacturer’s protocol.

Techniques: Injection, Staining, Immunofluorescence, Microscopy, Quantitation Assay, Incubation, Blocking Assay