Effect of migracin A on soft agar colony formation of ES-2 cells.

<p>The cells were incubated for 6 days with each chemical. *, P<0.001.</p

Inhibition of migration and invasion by IGF-1 receptor inhibitor Linsitinib and PI3K/Akt inhibitor LY294002.

<p>A. Effect of Linsitinib on the viability of ES-2 cells. The cells were incubated with Linsitinib for 24 h, and viability was assessed by MTT. B. Effect of LY294002 on the viability of ES-2 cells. The cells were incubated with LY294002 for 24 h, and viability was assessed by MTT. C. Inhibition of cellular migration. Migration was measured by wound healing assay. The cells were incubated for 24 h. *, P<0.05. **, P<0.01 (n = 3). D. Inhibition of cellular invasion. Invasion was measured by Matrigel chamber assay. The cells were incubated for 24 h. *, P<0.05. **, P<0.01 (n = 5).</p

Inhibition of migration and IGF-1 expression by migracin A in JHOC-5 cells.

<p>A. Effect on viability. The cells were incubated with migracin A for 24 h. B. Inhibition of migration in JHOC-5 cells. The cells were incubated for 24 h. *, P<0.05. **, P<0.01. ***, P<0.001 (n = 3). C. Increase of vasohibin-1. The cells were incubated with 1 μg/ml of migracin A, and each mRNA was measured by PCR. D. Inhibition of IGF-1 mRNA expression. The cells were incubated with indicated concentration of migracin A for 24 h, and the mRNA was measured by PCR.</p

Inhibition of capillary tube formation by migracin A.

<p>A. Effect of migracin A on viability of HUVEC. The cells were incubated for 24 h, and viability was assessed by MTT. B. Inhibition of VEGF-induced tube formation by migracin A in HUVEC. The cells were incubated for 4 h. *, P<0.05. **, P<0.01 (n = 4).</p

Relation between vasohibin-1 and IGF-1 expressions.

<p>A. Effect of IGF-1 knockdown on vasohibin-1 expression. B. Decrease of IGF-1 expression by the overexpression of vasohibin-1. C. Possible mechanism for inhibition of migration and invasion by migracin A.</p

Involvement of vasohibin-1 in the inhibition of migration by migracin A.

<p>A. Effect of migracin A on vasohibin mRNA expressions in ES-2 cells. The cells were incubated with 1 μg/ml of migracin A, and each mRNA was measured by PCR. B. Over expression of V5-tagged vasohibin-1 in ES-2 cells. C. Inhibition of cellular migration by overexpression of vasohibin-1. D. Effect of vasohibin-1 overexpression on cellular invasion. *, P<0.01 (n = 3–6).</p

Inhibition of cellular migration and invasion by migracin A.

<p>A. Structures of migracin A and B. B. Effect of migracin A on the viability of ES-2 cells. The cells were incubated with migracin A for 24 h, and viability was assessed by MTT. C. Inhibition of cellular migration. Migration was measured by wound healing assay. The cells were incubated for 24 h. D. Inhibition of cellular invasion. Invasion was measured by Matrigel chamber assay. The cells were incubated for 24 h. *, P<0.05. **, P<0.01 (n = 3).</p

Involvement of IGF-1 in the inhibition of migration and invasion by migracin A.

<p>A. Inhibition of IGF-1 mRNA expression in ES-2 cells. The cells were incubated with indicated concentration of migracin A for 24 h, and the mRNA was measured by PCR. B. Knockdown of IGF-1 by siRNA in ES-2 cells. C. Inhibition of cellular migration by knockdown of IGF-1. D. Inhibition of cellular invasion by knockdown of IGF-1.</p

A proposed model of rapamycin’s neuroprotective effect during retinal inflammation.

<p>The basal level of activated mTOR promoted NF-κB activation during inflammation, which induced the retinal expression of the inflammatory cytokines, interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). Activated NF-κB elevated the activated and phosphorylated mTOR (pmTOR) level during inflammation, which may have exacerbated the pathogenesis. The subsequent retinal activation of signal transducer and activator of transcription 3 (STAT3), at least partly, reduced the rhodopsin protein expression in a post-transcriptional manner and impaired rod photoreceptor function. Rapamycin also attenuated inflammatory leukocyte adhesion to the vessel walls, and increased potentially neuroprotective autophagy in the retina during inflammation.</p

The interaction between mTOR and NF-κB in the retina during inflammation.

<p>Immunoblot analyses (A-H). The protein level of phosphorylated and activated mTOR (pmTOR) was not increased in the retina 3 hours after the induction of inflammation by an intraperitoneal lipopolysaccharide (LPS) injection, although the level of pmTOR was suppressed by the rapamycin treatment (A, B). At 12 hours after the LPS injection, the pmTOR expression was increased in the retina, which was suppressed by the rapamycin treatment (C, D). The pmTOR level in the retina at 12 hours was also suppressed by the NF-κB inhibitor, DHMEQ (E, F). The ratio of the LC3II to LC3I protein levels (an autophagy marker) in the retina was not changed after LPS injection but was increased by rapamycin at 12 hours after LPS injection (G, H); n = 5. *p < 0.05, **p < 0.01.</p