Figure 4. Enhanced chemotaxis of macrophages and angiogenesis by CHI3L1-induced IL-8 and MCP-1 protein secretions.

(A) The protein secretions of IL-8 and MCP-1 in the supernatant assessed by ELISA. CHI3L1 transfection (CH) in SW480 cells for 48 hrs significantly and dose-dependently increased the protein secretion of IL-8 and MCP-1 as compared with empty vector (EV) transfection. Stimulation with CHI3L1 (80 ng/ml) in SW480 cells significantly increased the protein secretion of IL-8 and MCP-1.

(B) The mRNA expressions of IL-8 and MCP-1 determined by quantitative RT-PCR. The mRNA levels of IL-8/GAPDH and MCP-1/GAPDH were significantly increased in CHI3L1 overexpression (CH) as compared with those in empty vector (EV) transfection.

(C) The protein secretions of IL-8 and MCP-1 in the supernatant assessed by ELISA. Two kinds of CHI3L1 miRNAs (miRNA 1, miRNA 2) transfection in SW480 cells for 48 hrs significantly decreased the protein secretions of IL-8 and MCP-1 as compared with negative control miRNA (NC) transfection.

(D) Mean numbers of migrated THP-1 cells at x200 magnification. Incubation with IL-8 or MCP-1 neutralizing Ab significantly diminished the migration of THP-1 cells induced by CHI3L1 in SW480 cells as compared with control goat IgG.

(E) Mean numbers of migrated HUVECs at x200 magnification. Incubation with IL-8 or MCP-1 neutralizing Ab significantly diminished the migration of HUVECs induced by CHI3L1 in SW480 cells as compared with control goat IgG.

(F) Mean numbers of tube-forming structures at x100 magnification. Addition of IL-8 or MCP-1neutralizing Ab during preparation of conditioned medium from empty vector-transfected or CHI3L1-overexpressed SW480 cells significantly inhibited the tube formation of HUVECs as compared with control goat IgG.

All results (A-F) are the mean (± SEM) of more than three separate experiments. * P < 0.05, ** P < 0.01.