SupplementalFigure S1. ThemRNA levels of Wnts and Frizzleds (FZ) inthe retina of STZ-induced diabetic and OIR rats. Equal amounts of mRNA from the retinas ofSTZ-induced diabetic rats 16 weeks after the onset of diabetes and age-matched non-diabetic rats (A&B), and OIR rats and normal rats at age of P16 (C&D) were used for real-time RT-PCR and normalized by 18s rRNA levels. Primer sets specific for Wnts and FZ,which are known to be expressed in the retina and in endothelial cells, were used for the PCR. The retinal mRNA levels of the Wnts and the Fz in the STZ rats and OIR rats were expressed as ratios to their matched normal control levels (mean ± SD, n = 7).
Real-time reverse transcription (RT)-PCR: Total RNA was isolated from freshly dissected retina using a commercial kit (Qiagen, SantaClarita, CA). Primers for Wnts and Frizzled(Fz) receptors were designed from the cDNAsequences spanning an intron which is longer than 1 kb using Primer3 software( The primer sequences were presented in Supplemental Table 1. Total RNA (1.0 µg) was used for RT reaction, and 1 µl of the RT productwere used for real-time PCR with a SYBR Green PCR Master Mix (Applied Biosystems) as described previously.
Supplemental Figure S2. Constitutive β-catenin expression up-regulates VEGF levels. To determine the impacts of the Wnt signaling activation, we also over-expressed a constitutively active mutant of β-catenin, in which a phosphorylation site (S37) was replaced by Ala, using an adenovirus vector (Ad-S37A). Primary human retinal capillary endothelial cells (HRCEC), human pericytes, and macrophages were infected with Ad-S37A, at MOI of 20, with the same titer of control virus (Ad-GFP) as control. As shown by Western blot analysis, expression levels of VEGF and total β-catenin levels were elevated in the cells infected by Ad-S37A, compared with that infected by the control virus.