Journal: International journal of cancer

Article Title: Interleukin-34 promotes tumor progression and metastatic process in osteosarcoma through induction of angiogenesis and macrophage recruitment.

doi: 10.1002/ijc.29376

Figure Lengend Snippet: Figure 3. The cell signalling induced by IL-34 in endothelial cells depends on cell surface glycosaminoglycans. Human ECFCs (a) and HUVECs (b–c) were treated with 200 ng/mL of IL-34 or M-CSF in the presence or absence of 5 ng/mL FGF-2 for 1–15 min (a, b) or for 10 min (c). FAK, Akt, Src, ERK1/2, and p38 phosphorylations were analyzed by Western blot compared to the levels of GAPDH. (d) ECFCs were treated with 50 ng/mL of IL-34 in the presence of 5 ng/mL FGF-2 and 10 lM of signalling pathway inhibitors (PF 573228, a FAK inhibitor; PP2, a Src inhibitor; Wortmannin, a PI3K/Akt pathway inhibitor; UO126, an ERK1/2 inhibitor. ECFCs were then seeded on Matrigel V R in growth factor-depleted basal medium. After 18 h of culture the cells were fixed and stained with Giemsa. After 18 h of culture, comparison of the mean (6 SEM) total length of tubules (% of control ECFCs) formed in three independent experiments. Results are normalized to untreated ECFCs. (e) ECFCs were pretreated for 2 h at 37C with a mixture of 0.5 U/mL heparinase I, II and III and 0.2 U/mL chondroitinase ABC, for 30 min before 50 ng/mL IL-34 stimulation. Light-micrographs show the typical appearance of tubules formed by IL-34 pretreated ECFCs in Matrigel V R, markedly reduced after enzymatic treatment (original magnification: 43). (f) Similar enzymatic treatment as described above abolishes the IL-34-induced signalling pathways in HUVECs. *p < 0.05, **p < 0.01 compared to the control condition (in absence of FGF2 and IL-34). ##p < 0.001 compared to the FGF21 IL-34 condition).

Article Snippet: The membranes were blotted with the antibodies (1/1,000) anti-pFAK (Tyr925), anti-pAkt (Ser473), anti-pSrc (Tyr416), anti-p38 (Thr180/Tyr182), antipERK 1/2 (Thr202/Tyr204), or with antibodies against the total forms of proteins above (Cell Signalling, Danvers, MA).

Techniques: Western Blot, Staining, Comparison, Control