Fig. 5

Collagen triple helix repeat containing 1 (CTHRC1) effects transwell cell migration. (a) Western immunoblotting detection of CTHRC1 in rheumatoid arthritis-fibroblast-like synoviocytes (RA-FLS), NIH 3T3, C3H/10 T1/2. RA-FLS express CTHRC1, but murine fibroblasts are negative for CTHRC1. (b) RA-FLS also express cadherin CDH11, alpha smooth muscle actin (α-SMA), and α-tubulin. Staining membrane with secondary antibodies alone was negative. (c) CTHRC1 is secreted into conditioned media upon transfection and overexpression in Chinese hamster ovary (CHO) cells. Lanes: (1) positive control 10 ng rhCTHRC1; (2) day 0; (3) day 1; (4) day 3; (5) day 5. (d) RA-FLS were treated directly in transwell chambers with rhCTHRC1 protein, while FBS concentration was varied from 0 %, 1–6 %. Cells that migrated through the membrane were stained with toluidine blue and counted under the microscope. Solid black columns represent cells treated with rhCTHRC1, gray columns are untreated cells. (e) murine C3H/10T1/2 fibroblasts were treated with rhCTHRC1 similarly to RA-FLS. (f) Serum from mice with acute CAIA (5 % serum mixed with 5 % FBS in complete media) showed the strongest promigratory effect. (g) Toluidine blue staining and microscopy of cells migrated through the membrane and spread on its opposite side after the completion of the experiment. Numerous dots are membrane pores of 8 μm in size. Data are representative of three similar but independent experiments. Statistical significance was calculated using Student’s t test: *p < 0.05; **p < 0.01 for treatment versus no treatment comparison