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25?mg/animal) was dissolved in 20?��l of 100% ethanol. For i.p. delivery, 9-cis RAL was dissolved in 200?��l vehicle (10% ethanol/10% BSA in 0.9% NaCl) and injected into the peritoneal cavity. Retinoid was handled under a dim red light. All procedures were performed under a dim red light on dark-adapted animals using methods modified from those previously described (Tang et?al., 2011). For each sample, two eyecups from 20-week-old Atg5��RPE and Atg5f/f littermate controls were pooled after hemisection and the removal of the anterior segment. Data were then averaged for each group of Atg5��RPE (n?= 4) and Atg5f/f (n?= 5). See Extended Experimental Procedures for details. All data were analyzed using a two-tailed, unpaired Student��s t test. Statistical significance was considered as p?allobarbital for histology, TEM, reagents, and antibodies are available in the Extended Experimental Procedures. Extended Experimental Procedures BEST1(VMD2)-cre transgenic mice, in which cre recombinase is controlled by a fragment of the human VMD2 promoter (Apte et?al., 2006?and?Esumi et?al., 2004), were generated by established protocols in the Department of Ophthalmology and Visual Molecular Genetics Core laboratory. Crossing this line to the Atg5flox/flox (generously provided by Dr. Noboru JNK inhibitor mw Mizushima, Tokyo Medicine and Dental University, Tokyo, Japan) (Hara et?al., 2006) generated the Atg5��RPE mouse line in which Atg5 was specifically deleted in RPE cells. Atg5��RPE offspring are born viable, in reasonable numbers, and do not show any developmental defects in the eye or other organs at birth (not shown). The Atg5��RPE mouse strain AZD2014 concentration was also crossed to the GFP-LC3 transgenic mice (also provided by Dr. Mizushima) ( Mizushima et?al., 2004) to generate the Atg5��RPE-GFP-LC3 mouse strain. C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME) (stock number 000664). Ulk1?/? and Ulk1+/? mice were kindly provided by Dr. Mondira Kundu (St. Jude Children��s research Hospital, Memphis, TN). All transgenic and conditional knockout lines were backcrossed to C57BL/6J and verified as congenic with the C57BL/6J strain by microsatellite analysis (Research Animal Diagnostic and Investigative Laboratory, RADIL, University of Missouri, Columbia, MO). All strains were free of the rd8 mutation by PCR analysis ( Mattapallil et?al., 2012). Mice were housed under cyclic light conditions with lights on at 6:00 AM and off at 6:00 PM. In all experiments littermate control animals were used (Atg5f/+;cre+ or Atg5f/f). All experiments contained at least three mice per group and were repeated a minimum of three times. Chloroquine (CLQ) was purchased from Sigma-Aldrich (St. Louis, MO), 4��, 6-diamidino-2-phenylindole (DAPI) was purchased from Life Technologies, Grand Island, NY).