Figure 2

CTHRC1 acts through the ERK signaling cascade to induce Ang-2 expression in ECs. (a) Gene expression in rCTHRC1-treated HUVECs was analyzed by RT-qPCR. Differentially expressed genes between the rCTHRC1 and vehicle groups are reported as the mean±s.d. (*P<0.01 versus vehicle). (b) HUVECs were treated with rCTHRC1 (0.5 μg ml⁻¹), and Ang-2 secreted into the culture supernatant was measured by ELISA. Data are reported as the mean±s.d. (**P<0.001 versus vehicle). (c) HUVECs were stimulated with rCTHRC1 (0.5 μg ml⁻¹) for the indicated times. Whole-cell lysates were prepared and used for Western blotting with antibodies against ERK, p-ERK and actin. Representative images from three independent experiments are shown. (d) HUVECs were co-stimulated with the ERK inhibitor PD98059 (100 μM) and rCTHRC1 (0.5 μg ml⁻¹) for 18 h, and secreted Ang-2 in the culture supernatants was measured by ELISA. (e) Nuclear fractions were collected from HUVECs treated with rCTHRC1 for the indicated times, after which c-fos, p-c-fos and lamin (positive control for the nuclear fraction) were detected by western blotting. Representative images from three independent experiments are shown. (f) HUVECs were transfected with AP-1-luc and pRLnull and treated with rCTHRC1 for the indicated times. Dual luciferase activities were determined after treatment, as described in the Materials and Methods section. The data represent firefly luciferase activity normalized to Renilla luciferase activity present in each sample. Data are reported as the mean±s.d. (*P<0.01, ^#P<0.05, **P<0.001 versus vehicle). CTHRC1, collagen triple-helix repeat-containing 1; EC, endothelial cell; ELISA, enzyme-linked immunosorbent assay; rCTHRC1, recombinant CTHRC1; RT-qPCR, reverse transcription quantitative PCR.