AICAR suppressed tumor angiogenesis and inflammatory cells infiltration.

<p>(A, B) Microvessel density in tumor tissues was determined by immunofluorescent staining by an endothelial-specific antibody CD31. (A) Control group and (B) AICAR-treated group. (C) Quantitative analysis of fluorescent-positive area (per 4000 µm²) in tumors. Vessel density was significantly suppressed in AICAR-treated mice group (**p<0.01). (D, E) Macrophage- and neutrophil- infiltration in Y79 xenografts. Typical photomicrographs of immunofluorescent staining for CD11b (red) in Y79 xenografts. Nuclei were stained with propidium iodide (blue). Y79 cells isolated from control mice (D) and AICAR-treated mice (E). (F) Quantitative analysis of the CD11b (+) cells/DAPI (+) cells ratio in tumors. The number of CD11b (+) cells was significantly lower in the AICAR-treated mice group than in the control mice group (**p<0.01). Each column represents the mean ± SEM. Scale bars (A, B, D, E), 200 µm.</p

AICAR does not alter the levels of cyclins A, D and E in retinoblastoma in vivo.

<p>Quantitative RT-PCR analysis of tumors treated with AICAR in comparison with control shows no significant difference. Each column represents the mean ± SEM.</p

AICAR suppressed proliferation and induced apoptosis of retinoblastoma in vivo.

<p>(A, B) Immunofluorescent analysis for Ki67 of tumors of Y79 cells isolated from control mice (A) and AICAR-treated mice (B). Nuclei were stained with propidium iodide (red). (C) Quantitative analysis of Ki67 (+) cells/PI (+) cells ratio in tumors. Values are significantly lower in the AICAR-treated mice group than in the control mice group (**p<0.01). (D,E) Apoptotic cells in retinoblastoma xenografts. Typical photomicrographs of apoptotic cells using TUNEL assay (green) in Y79 xenografts. Nuclei were stained with propidium iodide (red). Y79 cells isolated from control mice (D) and AICAR-treated mice (E). (F) Quantitative analysis of the apoptotic cell percentage in tumors. Note that the number of TUNEL (+) cells was significantly higher in the AICAR-treated mice group than in the control mice group (**p<0.01). Each column represents the mean ± SEM. Scale bars (A, B, D, E), 200 µm.</p

Proposed mechanism of action for AICAR in human retinoblastoma in an in-vivo xenograft model.

<p>AICAR administration leads to activation of AMPK decreased tumor vessel density and decreased CD11b (macrophage) infiltration. Activated AMPK inhibits mTOR pathway, protein synthesis and cell growth. In addition, AICAR administration results in decreased levels of p21, which was recently found to inhibit apoptosis and promote cell proliferation. Overall signaling changes leads to loss of viability due to apoptosis, proliferation block and inhibition of tumor progression.</p

AICAR treatment of retinoblastoma is associated with activation of AMPK, inhibition of mTORC1 and decrease of p21.

<p><b>A.</b> AICAR treatment of retinoblastoma is associated with activation of AMPK. Western blot analysis of phosphorylated ACC (Ser-79) (a downstream effector of AMPK) showed significant increase of pACC in tumours from AICAR treated mice comparing to control (**p<0.01, n = 19). <b>B and C.</b> Treatment with AICAR resulted in the inhibition of the mTORC1 pathway. Western blot analysis of tumor xenografts harvested from mice treated with AICAR showed significant decrease of mTOR pathway downstreams, pS6RP (Ser235/236) and the p4E-BP1 (Ser65) when compared to PBS-treated mice (***p<0.001 for both, n = 17 for pS6RP and n = 23 for p4EBP1). <b>D.</b> AICAR down-regulates p21WAF1/Cip1 in AICAR treated tumors as shown via Western blot analysis (*p<0.05, n = 23). Density values bands are graphically expressed relative to control. GAPDH was used as a loading control in all panels. Multiple bands represent separate biological samples. Each column represents the mean ± SEM.</p

EGFL7 knockdown does not influence VEGFR2 phosphorylation or neuropilin 1 expression.

<p><i>A,</i> Mouse eye cups of each group were treated with EGFL7 or control siRNA after embedding them in Matrigel. Samples were cultured in VEGF (25 ng/ml) containing medium. At 3 days after knockdown of EGFL7, endothelial cells were collected using anti-mouse CD31 antibody-coated magnetic beads (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091849#pone.0091849.s004" target="_blank">Figure S4</a>). The amounts of p-VEGFR2, neuropilin 1 and EGFL7 were examined by Western blotting. <i>B,</i> Densitometry of p-VEGFR2 in panel A. <i>C,</i> Densitometry of neuropilin 1 in panel A. <i>D,</i> Densitometry of EGFL7 in panel A. <i>ANOVA</i> Statistical analysis performed. (n = 3) <i>*, P<0.01. **, P<0.05.</i> NS, not significant. <i>*, P<0.01.</i> NS, not significant.</p

VEGF-induced tube formation is EGFL7 dependent.

<p><i>A,</i> Mouse eye cups of each group were treated with EGFL7 or control siRNA after embedding them in Matrigel. Samples were cultured in VEGF (25 ng/ml) containing medium. At 3 and 5 days after knockdown of EGFL7, the tube length of neovascular from samples was evaluated by immunofluorescence using CD31 antibody. Bar equals 1000 μm. <i>B, ANOVA</i> Statistical analysis performed to evaluate the area of tube length. (n = 6) <i>*, P<0.01. **, P<0.05.</i> NS, not significant. C. At 3, 5 and 7 days after knockdown of EGFL7, endothelial cells were collected using anti-mouse CD31 antibody-coated magnetic beads (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091849#pone.0091849.s004" target="_blank">Figure S4</a>). The amounts of EGFL7 in isolated cells were examined by Western blotting.</p