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: Immortalized splenic macrophage cell line I-13.35 (CRL-2471, ATCC) derived from TLR4-defective C3H/HeJ strain and human 18Co colonic fibroblasts (CCL-228, ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 20% LADMAC conditioned media and Eagle’s minimum essential medium (EMEM), respectively, supplemented with 10% FBS (Hyclone) at 37°C with 10% CO 2 .

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