Supplementary figure 1. Metformin inhibits the mTORC1 signaling pathway before the initiation of endothelial cell degeneration.Metformin (200 mg/kg) or the vehicle (saline) was administered to P4 normal mice, and their eyes were collected 6 h later. A: Representative images of retinal flat mounts stained for PECAM-1 and phosphorylated S6 protein (pS6). Scale bar: 100 μm in A(a) (applies to A[b]-[h]). Metformin selectively decreased the pS6 immunoreactivity associated with vascular cells without affecting the vascular area density. B, C: Quantitation of vascular area density (B) and colocalization of vascular endothelial cells and pS6 (C). To quantify the colocalization of vascular endothelial cells and pS6, the surface area of where the pS6-positive pixels and the PECAM-1-positive pixels were colocalized was divided by the area of PECAM-1-positive pixels. Each column with a vertical bar represents the mean ± SE from 4 animals, *P < 0.05 versus Saline.