*2.1. Experimental Animals*

All animal procedures were performed in accordance with a) protocols approved by the Institutional Animal Care and Use Committees at University of Florida, Gainesville, FL, USA (#CR-201106001), Michigan State University, Lansing, MI, USA (#Busik09/14-160-00, and Indiana University, Indianapolis, IN, USA (#10574 MD/R), b, the National Institutes of Health Guide for Care and Use of Laboratory Animals and c) the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Male C57BL/6J mice were purchased from Jackson Labs at 6 weeks of age and male BBZDR/wor T2D rats and lean heterozygote nondiabetic control littermates were obtained from biomedical research models (BRM, Inc.) Worcester, Massachusetts at 5 months of age. C57BL/6J mice (n = 240) and BBZ rats (n = 75) were maintained on a standard 12/12 h light/dark cycle (6.00 am lights on/6.00 pm lights o ff). T1D was induced in eight-week-old C57BL/6 mice (n = 120) by five consecutive intraperitoneal injection of freshly prepared streptozotocin (STZ) solution with the concentration of 40mg/kg body weight in 0.1M/citrate bu ffer, pH 4.5 as previously described [**? ?** ]. The control group received sodium citrate bu ffer (vehicle) alone. Diabetes was confirmed by two consecutive blood glucose levels >240 mg/dl, using the AlphaTrak ® blood glucose monitor and test strip system (Abbot laboratories, Irvine, CA, USA), according to the manufacturer's instruction. No animal used in this study required insulin injections. Eyes from one group were collected at 2 months following establishment of diabetes and the second cohort 9 months after diabetes induction (the age of the animals at these time points being 4 and 11 months, respectively). We also investigated inbred Bio-Breeding Zucker (BBZDR)/wor T2D rats (n = 37) and age-matched controls (n = 38) [**?** ]. BBZDR/wor rats spontaneously became diabetic at ~10 weeks of age and were maintained for 4 months after the onset of diabetes. A second group of C57BL/6J mice (n = 24) were dark adapted for 48 h before sacrifice and referred to as being on the dark/dark cycle. In all groups, animals were euthanized by deep anesthesia with isoflurane followed by cervical dislocation and eyes were immediately enucleated every 2 or 3 h over a 24 h period and prepared for immunofluorescence staining. For animals in the dark cycle, euthanasia and enucleation were performed in a dark room under a red safe light. For each set of experiments, all animals were started at the same time point to clearly identify any phase shift in peak or trough of ATG expression. The number of biological replicates of the enucleated eyes at each time point is n = 10 for mice and n = 3 for rats. Only one eye from each animal was used in the assessment.
